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. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: Methods Mol Biol. 2022;2374:121–137. doi: 10.1007/978-1-0716-1701-4_11

Characterization of Smoothened Phosphorylation and Activation

Jianhang Jia 1, Yuhong Han 2, Yong Suk Cho 3, Jin Jiang 4
PMCID: PMC8941978  NIHMSID: NIHMS1786394  PMID: 34562248

Abstract

The GPCR-family protein Smoothened (Smo) is an obligatory signal transducer of the Hedgehog (Hh) signaling pathway. Binding of Hh to its receptor Patched (Ptc) alleviates Ptc-mediated inhibition of Smo, allowing Smo to activate the Cubitus interruptus (Ci)/Gli family of zinc finger transcription factors. The activation of Smo is an early and crucial event in Hh signal transduction. Studies have shown that Hh induces cell surface/ciliary accumulation and phosphorylation of Smo by multiple kinases, including protein kinase A (PKA), casein kinase 1 (CK1), casein kinase 2 (CK2), G protein-coupled receptor kinase 2 (Gprk2/GRK2), and atypical PKC (aPKC). Here, we describe the assays used to examine the phosphorylation and activity of Smo, including in vitro kinase assay, phospho-specific antibodies, luciferase reporter assay, cell surface accumulation, and ciliary localization assays. These assays provide powerful tools to study Smo phosphorylation and activation, leading to mechanistic insight into Smo regulation.

Keywords: Smoothened, Phosphorylation, Hedgehog, PKA, CK1, GRK2, CK2, aPKC, Primary cilium

1. Introduction

Hedgehog (hh) was originally discovered as a segment polarity gene involved in Drosophila embryo development [1]. The Hh family of secreted proteins plays critical roles in cell growth and patterning in species ranging from insects to mammals; not surprisingly, misregulation of Hh signaling has been implicated in a wide range of human disorders including birth defects and cancer [2, 3]. The Hh signal is transduced by a largely conserved signaling cascade among different species [3, 4]. Smoothened (Smo), a G protein-coupled receptor (GPCR) family member, is essential for transducing the Hh signal in both insects and vertebrates [5]. Abnormal activation of Smo results in basal cell carcinoma and medulloblastoma, making Smo an attractive therapeutic target. Indeed, several Smo inhibitors including vismodegib have been approved by FDA for treating advanced basal cell carcinoma (BCC). However, cancer cells can acquire resistance through mutations in Smo and other mechanisms [6, 7]. Therefore, a better understanding of the mechanisms of Smo regulation in development and homeostasis is critical for developing more effective therapeutic treatments for cancers caused by Smo dysregulation.

Studies in both Drosophila and mammalian cells have shown that Hh induces phosphorylation of Smo by multiple kinases, including protein kinase A (PKA; Drosophila only) and casein kinase 1 (CK1), casein kinase 2 (CK2), atypical PKC (aPKC), and G protein-coupled receptor kinase 2 (Gprk2/GRK2), which activate Smo by inducing its cell surface accumulation (Drosophila)/ciliary localization (vertebrates) and conformational change in Smo C-terminal intracellular tail [8–18]. Identification of phosphorylation sites on Smo by PKA, CK1, and Gprk2/GRK2 has relied mainly on a combination of in vitro kinase assay, and in vivo functional assay of Smo variants with the identified sites mutated. Phosphorylation at individual sites in vivo in response to Hh has been determined by generating phospho-specific antibodies and phospho-tag gel mobility shift assay [14, 19, 20]. The luciferase reporter assay is widely used as a tool to study gene expression at the transcriptional level because it is convenient, relatively inexpensive, and provides quantitative measurements of pathway activity.

In this chapter, we discuss several methodologies to study the phosphorylation and activity of Smo in Drosophila and in mammalian cultured cells. We will detail how to: culture and transfect S2 cells and NIH/3T3 cells, evaluate Smo activation using luciferase reporters, perform in vitro and in vivo phosphorylation assays, and examine Smo subcellular localization by immunostaining.

2. Materials

All solutions are prepared using ultrapure water (purified by Milli-Q integral water purification system). All chemicals are analytical-grade or molecular biology-grade reagents.

2.1. Cell Culture and Transfection

  1. Drosophila Schneider 2 cells (S2 cells).

  2. S2 cell culture medium: Schneider’s Drosophila Medium with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin.

  3. Hh-conditioned medium: obtained from Hh stable cell line of S2 cells after 24 h induction with 0.7 mM CuSO4.

  4. Effectene transfection reagent.

  5. Transfection-grade plasmids: Ub-Gal4 and desired expression constructs cloned into pUAST vectors.

  6. Cell culture incubator at 25 °C.

  7. NIH/3T3 cells.

  8. NIH/3T3 cell culture medium: DMEM Medium with 10% bovine calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. (see Note 1).

  9. NIH/3T3 cell starving medium: DMEM medium with 0.5% bovine calf serum.

  10. Cell culture incubator at 37 °C.

  11. Polyjet in vitro DNA transfection reagent.

  12. Transfection-grade plasmids: desired protein coding sequences were subcloned into pGE and pcDNA3.1(+) vectors.

2.2. Luciferase Reporter Assay

  1. Transfection-grade plasmids: ptc-luciferase and Renilla reporter constructs, Ub-Gal4, tub-Ci (Ci under the α-tubulin promoter), and the desired Smo constructs. For mammalian cells-based assay, 8XGli1-Luciferase reporter, PRL-SV40 Renilla vector along with certain mSmo constructs were transfected.

  2. Firefly (Photinus pyralis) and Renilla (Renilla reniformis or sea pansy) luciferases detection kit, such as Dual-Luciferase Reporter Assay System.

  3. GLOMAX Multi Detection System Luminometer.

  4. Rocker.

  5. POLARstar OPTIMA plate reader.

2.3. Phospho-Specific Antibody Production

  1. The anti-SmoP antibody, which specifically detects Drosophila Smo phosphorylation at the second phosphorylation cluster, was generated by Genemed Synthesis Inc. by injecting the antigen peptide CRHVSVESRRN(pS)VD(pS)QV(pS)VK into rabbits. The serum was affinity-purified with the antigen and the flow-through was kept as a control antibody against nonphosphorylated peptide.

  2. The PS1 antibody was generated by Genemed Synthesis Inc. to detect mSmo phosphorylation at S1 site. The phosphorylated peptide EP(pS)ADV(pSpS)AWAQHVTC was injected into rabbits. The serum was affinity-purified by antigen and the flow-through from the affinity-purification was also kept as control antibody against nonphosphorylated peptide.

2.4. GST-Smo Fusion Protein Expression and Purification

  1. Escherichia coli BL21 (DE3) strain.

  2. E. coli culture medium: Luria-Bertani (LB) medium with 100 μg/mL ampicillin.

  3. 0.1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) for inducing fusion protein expression.

  4. Phosphate-buffered saline (PBS): 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 (pH 7.4).

  5. 2xPre-TGEM buffer and extraction buffer: prepare according to Tables 1 and 2. 2xPre-TGEM buffer is autoclaved and stored at 4 °C, but extraction buffer is freshly prepared.

  6. Glutathione Sepharose 4B agarose beads.

  7. 10 mg/mL lysozyme.

  8. Elution buffer: 10 mM GSH in 50 mM Tris–HCl (pH 8.0).

  9. Amicon Ultra-4 centrifugal filter unit with Ultracel-30 membrane.

  10. Incubator shaker at 37 °C.

Table 1.

2xPre-TGEM buffer

Stock Stock concentration Volume Final concentration
Tris 8.0 1 M 8 mL 40 mM
Glycerol 80 mL 40%
EDTA 0.5 M 0.8 mL 2 mM
MgCl2 1 M 2 mL 10 mM
NP-40 0.4 mL 0.2%
H2o 108.8 mL
Total 200 mL
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Table 2.

Extraction buffer

Stock Stock concentration Volume Final concentration
Pre-TGEM 2x 50 mL
NP-40 0.9 mL 1%
NaCl 5 M 3 mL 150 nM
DDT 1 M 100 μL 0.1 M
PMSF 0.2 M 500 μL 1 mM
Benzonase nuclease 0.3 M 100 μL 300 nM
H2O 45.4 mL
Total 100 mL
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2.5. Kinase Assays

  1. Kinase:

    1. PKA kinase.

    2. CK1 kinase.

    3. Gprk2 kinase is purified by immunoprecipitation with the anti-Flag antibody using S2 cells transfected with Flag-Gprk2 or Flag-Gprk2 mutant.

    4. Recombinant human PKCζ.

  2. Kinase-specific reaction buffer (For PKA and CK1 kinase assays): 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2, and 5 mM DTT.

  3. Kinase-specific reaction buffer (For Gprk2 kinase assay): 20 mM Tris–HCl (pH 8.0), 2 mM EDTA, 10 mM MgCl2, and 1 mM DTT.

  4. Kinase-specific reaction buffer (For PKCζ kinase assay): 35 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 0.1 mM CaCl2, 0.5 mM EGTA, and 2.5 μM ATP or 10 μCi of γ-32P-ATP if needed.

  5. Phosphatase inhibitor: 50 nM okadaic acid (OA).

  6. Kinase inhibitors:

    1. PKC inhibitor: PKCζ pseudosubstrate.

    2. PKC inhibitor: Myristoylated.

    3. CK1 inhibitor: CK1-7 dihydrochloride.

    4. PKA inhibitor: H-89 dihydrochloride hydrate (see Note 2).

  7. Lysis buffer for cultured S2 cells: 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 1.5 mM EDTA, 10% glycerol, 1% NP-40, 10 mM NaF, 1 mM Na3VO4, and protease inhibitor tablet.

  8. Protein A ultralink resin.

  9. SDS Loading buffer (4x): 0.25 M Tris–HCl (pH 6.8), 8% SDS, 20% 2-mercaptoethanol, 40% glycerol, and 0.008% bromophenol blue.

  10. Running buffer: 25 mM Tris base, 190 mM glycine, 0.1% SDS.

  11. Transfer buffer: 25 mM Tris base, 190 mM glycine, 20% methanol.

  12. Molecular weight markers.

  13. Polyvinylidene difluoride (PVDF) membranes.

  14. TBST solution: 20 mM Tris–HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween-20.

  15. Blocking buffer: 5% nonfat dried milk or bovine serum albumin (BSA) in TBST.

  16. Chemiluminescence detection reagents: Immobilon western chemiluminescent HRP substrate, include luminol reagent and peroxide solution.

  17. Blue X-Ray Film.

  18. Stripping buffer: 62.5 mM Tris–HCl (pH 6.8), 100 mM 2-Mercaptoethanol, and 2% SDS.

  19. Antibodies:

    1. Rabbit anti-SmoP (1:10).

    2. Rabbit anti-PS1 (1:50).

    3. Mouse anti-SmoN (1:10).

    4. Rabbit anti-GST (1:10,000).

    5. Mouse anti-Flag M2 (1:1000).

    6. Mouse anti-Myc, 9E10 (1:50).

    7. Rabbit anti-GFP (1:1000; see Note 3).

    8. HRP-conjugated secondary antibodies.

  20. SDS-PAGE Gel.

2.6. Phospho-Tag Gel

  1. Resolving gel solution (0.375 mol/L Tris, 0.1 mmol/L MnCl2, 0.1% SDS). The following contents are based on the case of preparation of the 10 mL solution with 12 w/v% polyacrylamide gel and 50 μmol/L Phos-tag Acrylamide. Mix all contents well and gently pour into the gel caster—4 ml of 30% (w/v) acrylamide solution, 2.5 mL of 1.5 mol/L Tris/HCl pH 8.8, 0.10 mL of 5.0 mmol/L Phos-tag solution (NARD ltd), 0.10 mL of 10 mmol/L MnCl2, 0.01 mL of 10%(w/v) SDS solution, 50 μL of ammonium persulfate solution, 10 μL of TEMED (tetramethylethylenediamine), and 3.14 mL of distilled water (see Note 4).

  2. Stacking gel solution (0.125 mol/L Tris, 0.1% SDS). The following contents are based on the case of preparation of the 10 mL 4.5% polyacrylamide gel—1.5 mL of 30% (w/v) acrylamide solution, 2.5 mL of 0.50 mol/L Tris/HCl pH 6.8, 0.10 mL of 10%(w/v) SDS solution, 50 μL of ammonium persulfate solution, 10 μL of TEMED (tetramethylethylenediamine), and 5.84 mL of distilled water.

  3. SDS Running Buffer: 25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 8.3.

  4. SDS-loading buffer 6X: 375 mM Tris/HCl, 9% SDS, 50% glycerol, 0.03% bromophenol blue.

  5. Tris-Glycine-SDS transfer buffer: 25 mM Tris, 192 mM glycine, and 0.1% SDS, pH approx. 8.6.

  6. TBST washing buffer: 25 mM Tris, 0.15 M NaCl, 0.05% Tween-20, pH 7.6.

  7. Mini-PROTEAN Gel System.

  8. Mini Trans-Blot Electrophoretic Transfer System.

  9. Nitrocellulose membrane.

  10. 3 M Filter paper.

  11. Blocking buffer: 5% skim milk in TBST.

  12. Antibody incubation buffer: 1% skim milk in TBST.

  13. Enhanced chemiluminescent (ECL) detection reagent.

2.7. Cell Surface Staining of Drosophila S2 Cells

  1. 95% Ethanol.

  2. poly-L-lysine solution.

  3. Coverslips.

  4. Glass slides.

  5. 4% Formaldehyde prepared freshly from a 37% stock.

  6. Normal blocking sera: normal donkey serum or normal goat serum.

  7. PBST: PBS supplemented with 1% Triton X100.

  8. Antibodies:

    1. Mouse anti-SmoN (1:10).

    2. Epitope-tag.

  9. Secondary antibody: minimum cross-reactive (min X) secondary antibodies.

  10. Vectashield Mounting medium H-1000.

  11. Fingernail polish.

  12. Olympus Fluoview 1000 confocal microscope.

2.8. Cilium Staining of NIH/3T3 Cells Expressing Myc-Tagged mSmo

  1. Lab-Tek II CC2 Chamber slide system (8-well).

  2. Antibody: mouse anti-Acetylated tubulin (1:1000).

  3. Antibody: rabbit anti-Myc (1:1000).

  4. 4% PFA was freshly prepared from 16% stock solution from Electron Microscope Science.

  5. NIH/3T3 cells transduced with lentivirus expressing N-terminal 6XMyc-tagged mSmo (FUXW Myc Smo).

  6. Recombinant human Sonic Hedgehog N-terminus protein.

  7. Smoothened agonist SAG.

  8. PBS supplemented with 1% BSA.

  9. DAPI.

3. Methods

3.1. Luciferase Reporter Assay

3.1.1. S2 Cells

Drosophila Schneider 2 cells (S2 cells) are one of the most commonly used Drosophila cell lines. S2 cells were derived from a primary culture of late stage Drosophila embryos and are commercialized by several vendors.

  1. Seed 2 × 105 S2 cells per well in a 48-well plate (in 250 μL S2 cell culture medium) and let cells attach and grow for 16 h in a humidified incubator at 25 °C.

  2. Transfect the cells using Effectene transfection reagent according to the manufacturer’s instructions. Briefly, prepare the transfection mixture in an Eppendorf tube: 100 μL buffer EC, 5 ng Renilla, 150 ng Ub-Gal4, 50 ng tub-Ci (see Note 5), and 150 ng ptc-luciferase reporter constructs with or without pUAST-Smo constructs (see Note 6). Then add 4 μL Enhancer and mix by vortexing. Incubate at room temperature for 5 min.

  3. Add 6 μL Effectene transfection reagent to the mixture. Mix thoroughly and incubate for another 15 min.

  4. Add the mixture onto the cells and gently swirl the plate to distribute the transfection complex.

  5. Incubate for 24 h, then replace culture medium for fresh S2 cell culture medium (negative control) or S2 medium containing 60% Hh-conditioned medium. Incubate for 24 h more.

  6. 48 h Post-transfection, lyse the cells for luciferase activity analysis using the Dual-Luciferase Reporter Assay System. Carefully remove the cell culture medium from the culture plate and rinse S2 cells with PBS. Then add 60 μL passive lysis buffer per well and gently shake the culture plate for 20 min at room temperature.

  7. Measure the signal of the two luciferases using a GLOMAX Multi Detection System Luminometer in a 96-well plate, with 20 μL lysate in each well.

  8. Set the machine to dispense 100 μL Luciferase Assay Substrate to measure firefly luciferase activity, and then dispense 100 μL Stop & Glo reagent to measure Renilla luciferase activity. Renilla luciferase activity will be used to normalize the ptc-luciferase activity.

  9. Each sample should be tested at least in quadruplicate and a representative assay should come from three independent experiments.

3.1.2. NIH/3T3 Cells

  1. Seed one million NIH/3T3 cells into each well of a 6-well plate.

  2. After 24 h, transfect cells with Polyjet in vitro DNA transfection reagents using 1:3 ratio (DNA: Polyjet). The plasmid transfected for each well includes: 0.5 μg of 8XGli1-Luciferase reporter, 100 ng of PRL-SV40 Renilla vector along with 0.5 μg of certain mSmo constructs.

  3. 48 h after the transfection, wash the attached cells once with PBS and lyse with 200 μL of 1X passive lysis buffer with agitation for 10 min at room temperature.

  4. Measure of luciferase activity using Dual-luciferase reporter assay system and a POLARstar OPTIMA plate reader. Add 20 μL of the above cell lysate into each well of 96-well plate.

  5. Use 75 μL of luciferase assay substrate and Stop & Glow reagent to measure the firefly and Renilla luciferase activity, separately.

3.2. In Vitro Kinase Assay

3.2.1. Preparation of GST-Smo C-Tail Fusion

  1. Inoculate single colony of glutathione S-transferase (GST)-Smo aa656–755 in E. coli BL21 (DE3) into E. coli culture medium with 100 μg/mL ampicillin. Incubate overnight at 37 °C in a shaker at 200 rpm.

  2. Dilute the culture 1:100 into the fresh E. coli culture medium and grow until the culture OD600 value reaches 0.5–0.6. Add 0.1 mM IPTG and continue to incubate at 30 °C for 3–4 h (see Note 7).

  3. Pellet the cells at 1500 ×g at 4 °C, then resuspend cells in 15 mL of extraction buffer containing 5 μg/mL lysozyme and incubate on ice for 30 min. All steps should be performed on ice or at 4 °C from here on.

  4. Sonicate the suspension until it becomes clear, at 35% output, alternating 8 s on/8 s off (16 min for a 15 mL sample). Increase length of time for larger samples.

  5. Spin for 10 min at 13,500 ×g at 4 °C and transfer the supernatant to a tube containing 500 μL pre-equilibrated Glutathione Sepharose 4B agarose beads per liter of bacterial culture. Rock at 4 °C overnight (see Note 8).

  6. Centrifuge at 100 ×g at 4 °C for 5 min, discard the supernatant, and wash the beads 3 times with chilled extraction buffer.

  7. After the last wash, remove the extraction buffer and add 500 μL of elution buffer. Rock at 4 °C for 30 min.

  8. Centrifuge the sample at 100 ×g at 4 °C for 5 min. Store the supernatant and repeat the elution for 4–5 times. Pool the eluates together and concentrate with centrifugal filter unit with Ultracel-30 membrane.

3.2.2. In Vitro Phosphorylation Assays

Recombinant of purified PKA, CK1, Gprk2 (purified from S2 cells), and PKC kinases is used to phosphorylate GST-Smo fusion proteins in vitro, followed by analysis by western blot or autoradiography (if γ-32P-ATP is used in the phosphorylation reaction). When using radioisotopes, make sure to shield the samples with a Plexiglas screen and to survey the work area immediately afterward.

  1. Incubate GST-Smo with individual kinases at 30 °C for 30 min in 50 μL of the kinase-specific reaction buffer. Stop the reaction by adding 4X SDS loading buffer to the sample.

  2. Boil the sample at 100 °C for 5 min and centrifuge at 13,500 × g in a microcentrifuge at 4 °C for 5 min. Samples can be aliquoted and stored at −20 °C to avoid repeat freeze/thaw cycles.

  3. Load equal amounts of protein into the wells of an SDS-PAGE gel, along with molecular weight markers, and run the gel for 1–2 h at 90 V.

  4. Transfer the protein from the gel to the PVDF membrane at 180 mA for 3 h at 4 °C (see Note 9). The gel should be on the cathode side and the membrane on the anode side of the transfer sandwich.

  5. Block the membrane for 1 h with blocking buffer, then incubate membrane with appropriate dilutions of anti-SmoP for 2 h at room temperature (see Note 10).

  6. Wash the membrane three times with TBST solution, 10 min each, followed by incubation with recommended dilution of labeled secondary antibody in blocking buffer at room temperature for 1 h.

  7. Wash the membrane three times with TBST solution, 10 min each, then mix equal volumes of luminol reagent and peroxide solution in a clean tube (see Note 11) and add it onto the membrane of the blot protein side.

  8. Incubate for 5 min and cover the membrane with a plastic wrap, drain the excess substrate, and remove the air bubbles.

  9. Put an X-ray film onto it in the autoradiography cassette for 1 min (see Note 12). The anti-SmoP antibody can recognize the phosphorylated forms of Smo.

  10. Submerge the membrane in stripping buffer and incubate at 50 °C for 30 min with occasional agitation to remove any bound antibodies, then wash the membrane for 10 min in TBST solution three times at room temperature (see Note 13).

  11. Re-block the membrane in blocking buffer for 1 h at room temperature and proceed with the standard western blot protocol if wanted, such as to evaluate total GST-Smo.

  12. Proceed to expose the membrane to a new X-ray film for autoradiography if γ-32P-ATP is used in the phosphorylation reaction. Expose at −80 °C for 24 h, and adjust further exposure based on the initial signal.

3.3. In Vivo Phosphorylation Assay

3.3.1. Detect Smo Phosphorylation in S2 Cells Using Anti-SmoP Antibody

  1. Seed 1 × 106 S2 cells per well in a 6-well plate using 2 mL S2 cell culture medium per well and let grow for 16 h in a 25 °C incubator. Add 18 μg double-stranded RNA (dsRNA) to the medium to treat S2 cells if depletion of an endogenous protein is desired.

  2. Transfect 350 ng attB-Myc-Smo and 150 ng Ub-Gal4 using Effectene transfection reagent like in Subheading 3.1, step 2. Any pUAST-Smo construct can be used in this assay.

  3. Incubate for 24 h, then change cell culture medium into either control medium or medium containing 60% Hh-conditioned medium for additional 24 h. Use kinase inhibitor treatment for S2 cells if needed (see Note 14).

  4. 48 h Post-transfection, harvest S2 cells and add 450 μL lysis buffer. Then obtain cell lysate by centrifuging at 13,500 ×g for 10 min at 4 °C and separating the supernatant.

  5. The lysate is subjected to immunoprecipitation (IP). Add primary antibody to the cell lysates and incubate at 4 °C for 2 h, then add 30 μL of protein A beads ultralink resin.

  6. Wash the resin with wash buffer 3 times for 5 min each on rocker at 4 °C. Then obtain 30 μL IP samples by centrifuging at 1, 200 ×g for 3 min at 4 °C, followed by adding 4X SDS loading buffer to the IP sample. 5 μL IP sample is loaded for each run.

  7. Carry out western blot with the method described in Subheading 3.2.2. Hh consistently induces Smo phosphorylation, which can be readily detected by the anti-SmoP antibody. For better distinguishing phosphorylated forms from the unphosphorylated form of Smo, run 8% separating gel until the 55 kDa marker is about to reach the bottom of the gel.

  8. Electrophoretic mobility shift can also be used to detect the Smo phosphorylation [19]. Different levels of Hh activity induce differential phosphorylation of Smo (see Note 15). The OA treatment results in peak levels of phosphorylation in mobility shift of the transfected Myc-Smo.

3.3.2. Detect Smo Phosphorylation in NIH/3T3 Cells Using Anti-PS1 Antibody

  1. Seed one million NIH/3T3 cells and let grow for 24 h before transfection.

  2. Transfect 1ug of Myc-mSmo and its mutants’ constructs using PolyJet in vitro DNA transfection reagent.

  3. The next day, starve the cells with NIH/3T3 cell starving medium for 12–16 h.

  4. Treat the cell with 0.1ug/ml recombinant Shh-N or 100 nM SAG for 12–16 h.

  5. Collect the cells and incubate with cold lysis buffer at 4 °C for 10 min.

  6. Remove the cell debris by centrifugation and load a small aliquot of lysate onto an SDS-PAGE.

  7. Carry out a regular western blot to detect the phosphorylation of Smo protein with anti-PS1 antibodies.

3.3.3. Detect Smo Phosphorylation in NIH/3T3 by Phospho-Tag Gel

  1. Myc-mSmo samples are prepared as in Subheading 3.3.2.

  2. Use Resolving Gel Solution and Stacking Gel Solution to prepare a phospho-tag gel.

  3. Remove and clean up any remaining gel debris in the wells. Load 20 μL of samples that are already mixed with 6x loading buffer into each well.

  4. After gel electrophoresis is complete, trim the wells from the gel and place the gel into a suitable tray containing 1X Tris-Glycine-SDS transfer buffer. Allow the gel to equilibrate for 15–20 min.

  5. Soak the nitrocellulose membrane and two sheets of extra-thick blotting filter paper in 1X Tris-Glycine-SDS transfer buffer. The membranes should be briefly in deionized water before soaking in transfer buffer.

  6. Prepare the gel-membrane sandwich and run transfer in membrane transfer assembly.

  7. After transfer, disassemble the gel sandwich and discard the polyacrylamide gel and filter paper sheets, and then wash the membrane with 1X TBST washing buffer.

  8. Block with blocking buffer for 1 h at room temperature.

  9. Dilute the primary antibody in 1% milk in TBST and incubate overnight at 4 °C.

  10. Wash at least 4 times with TBST for 15 min.

  11. Dilute the secondary antibody in Antibody incubation buffer and incubate for 1 h at room temperature.

  12. Wash at least 4 times with TBST washing buffer for 15 min.

  13. Detect chemiluminescent on X-Ray film with Enhanced chemiluminescent (ECL) detection reagent.

3.4. Cell Surface Staining Assay

  1. Sterilize the coverslips in 95% ethanol and air dry, then put them in a sterile petri dish containing 50 μg/mL poly-L-lysine solution and incubate for 1 h at room temperature (15–25 °C). Wash with sterile water and allow to air dry.

  2. Seed 1 × 106 S2 cells in 2 mL S2 cell culture medium per well in a 6-well plate containing one sterile cover slip in each well. Let grow cells for 16 h at 25 °C in a humidified incubator.

  3. Transfect 200 ng pUAST-CFP-Smo and 150 ng Ub-Gal4 using Effectene transfection reagent. 24 h after transfection, change medium into either 60% Hh-conditioned medium or control medium for 24 h.

  4. Aspirate medium from each well, and wash S2 cells with PBS 2 times for 10 min each, at room temperature.

  5. Follow by fixation with 4% formaldehyde in PBS for 20 min. Then wash cells with PBS 3 times for 10 min each.

  6. Add 1.5% normal blocking serum in PBS for 30 min to block nonspecific staining. Blocking serum ideally should be derived from the same species in which the secondary antibody is raised.

  7. Incubate the samples with the mouse anti-SmoN antibody (1:10) in 1% normal blocking serum in PBS for 2 h before cell permeabilization. The anti-SmoN antibody recognizes the extracellular domain of Smo [8].

  8. Rinse S2 cells with 1 mL PBST 2 times for 10 min each at room temperature to allow cell permeabilization. Then incubate cells with Rhodamine-conjugated goat anti-mouse IgG (1:500) in 1 mL PBST for 1 h in dark chamber.

  9. Wash three times with PBST for 10 min at room temperature in dark chamber.

  10. Remove cover slips from the 6-well plate and mount on slides with the cell side toward the mounting medium and seal the edges of the coverslip with fingernail polish.

  11. Immunofluorescence is analyzed using a confocal microscope, such as Olympus Fluoview 1000. Rhodamine signals represent the cell surface localized Smo and CFP signals represent the total Smo that is expressed. Quantification of cell surface and total Smo levels can be calculated using Metamorph software.

3.5. Ciliary Localization Assay

  1. Seed NIH/3T3 cells transduced with lentivirus expressing Myc-tagged Smo into chamber slide system (8-wells) and let grow to around 60–70% confluence (see Note 16).

  2. Replace the culture medium with NIH/3T3 cell starving medium and further incubated at 37 °C for 16–24 h (see Note 17).

  3. Add Recombinant Shh-N (0.1 μg/mL) or a Smo agonist, SAG (100 nM) and incubate cells for 24 h at 37 °C before the standard immunostaining procedure.

  4. Wash cells with PBS once and fix with 4% PFA in PBS for 30 min at room temperature.

  5. After wash with PBS, permeabilize cells with 0.1% Triton X-100 in PBS for 30 min at room temperature.

  6. Rinse cells with PBS and incubate with anti-Myc and anti-acetylated tubulin antibody in PBS supplemented with 1% BSA for 1–2 h at room temperature or overnight at 4 °C.

  7. After another round of PBS wash, incubate cells with appropriate fluorochrome-conjugated secondary antibodies alone with DAPI solubilized in PBS supplemented with 1% BSA for 1 h at room temperature.

  8. Mount the cells with mounting medium or 80% glycerol and cover with coverslip.

  9. Analyze the cells with Zeiss LSM 700 confocal microscope.

Table 3.

Kinase inhibitor

Kinase inhibitor Target kinase Final concentration Adding time before harvesting
PKCζ pseudosubstrate, PKC 10μM 4 h
myristoylated
CK1–7 dihydrochloride CK1 10μM 4 h
H-89 dihydrochloride hydrate PKA 10μM 4 h
Okadaic acid (OA) Phosphatase 50 nM 4 h
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Table 4.

Gel recommendations

Protein size (kDa) Gel percentage (%)
4–40 20
12–60 15
20–140 12.5
30–200 10
40–300 8
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Acknowledgments

We thank the members in Dr. Jia’s laboratory for detailed protocols. This work was supported by grants from the National Institutes of Health R35GM131807 to J. Jia and R35GM118063 to J. Jiang, and from Welch Foundation I-1603 to J.Jiang.

Footnotes

1

. 10% FBS can be used as a replacement, but for optimal growth of NIH/3T3 cells, BCS is preferred.

2

. The kinase inhibitors can be used to treat the S2 cells in cell medium, the effective conditions are in Table 3. For in vitro kinase assay, they can be added directly to the reaction buffer at proper concentration if needed.

3

. pUAST-GFP construct can be cotransfected into S2 cells and used as a transfection control using the GFP antibody.

4

. Phos-tag SDS-PAGE, where a polyacrylamide gel containing Phos-tag is used, can be prepared by adding Phos-tag Acryl-amide and MnCl2 in Resolving Gel when preparing SDS-PAGE gels. During migration, the phosphorylated proteins with the phosphate group bind to the divalent metal ions in Phos-tag gel, which decreases their migration speed leading to their separation from nonphosphorylated proteins. The contents of phosphorylation and the size of proteins are variable, so that appropriate concentrations of Phospho-tag solution with range from 20 to 100 μmol/L is recommended to be used as well as with various concentrations of acrylamide.

5

. Because S2 cells do not express Ci, tub-Ci needs to be cotransfected to evaluate the activity of Smo mutants on ptc-luciferase expression, which is driven by Ci binding to the ptc promoter region. Renilla is also transfected and its luciferase activity is used to normalize the ptc-luciferase activity to account for variations in cell transfection efficiency.

6

. The total amount of DNA transfected must be kept constant with the empty pUAST plasmid.

7

. Inducing expression with 0.1 mM IPTG at 30 °C for 3 h usually yields considerable amount of soluble protein, otherwise may form inclusion body aggregates. Lower temperature should be considered if the proteins are not soluble.

8

. Equilibrate the glutathione agarose beads with extraction buffer before loading supernatant onto them.

9

. The time and voltage may require some optimization, and gel percentage will depend on the size of the Smo fragment of interest (for a guide see Table 4).

10

. Incubating overnight at 4 °C is also advisable. Make sure that the buffer moves freely across the entire surface of the membrane.

11

. Approximately 100 μL of working HRP substrate is required per cm2 membrane area.

12

. For getting a clear signal, the appropriate duration time can be varied, the initial exposure time is 30 s. The chemiluminescent signal on the membrane will last 30 min, and fresh HRP substrate can be added to the same blot for consecutive exposures.

13

. The incubation can be performed at 70 °C or the incubation time increased to more stringent conditions. To verify the removal of the previous antibody, the membrane could be detected with the secondary antibody.

14

. When using phospho-specific antibodies to detect Hh-induced Smo phosphorylation in cultured cells, including an immunoprecipitation step to enrich Smo proteins with the tag antibody could enhance the signals detected by the phospho-specific antibodies.

15

. For showing the mobility shift of Smo, low voltage (about 90 V) and extended running time are recommended. The loading sample could be heated at 55 °C for 5 min to reduce Smo aggregation.

16

. It is noteworthy that cells with high or even full confluence could have better primary cilium staining and Hedgehog signaling response; however, full confluence can cause cell detachment from the bottom of chamber slide, which should be avoided.

17

. Certain protocol starves the cell with DMEM completely depleted with FBS/BCS for 24–48 h, which makes the cells extremely unhealthy.

Contributor Information

Jianhang Jia, Department of Molecular and Cellular Biochemistry, Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA.

Yuhong Han, Department of Molecular Biology, UT Southwestern Medical Center at Dallas, Dallas, TX, USA.

Yong Suk Cho, Department of Molecular Biology, UT Southwestern Medical Center at Dallas, Dallas, TX, USA.

Jin Jiang, Department of Molecular Biology, UT Southwestern Medical Center at Dallas, Dallas, TX, USA.

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