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. Author manuscript; available in PMC: 2019 Nov 15.
Published in final edited form as: Methods Mol Biol. 2019;1957:69–82. doi: 10.1007/978-1-4939-9158-7_4

Methods to Investigate β-Arrestin-Mediated Regulation of GPCR Function in Human Airway Smooth Muscle

Tonio Pera 1, Raymond B Penn 2
PMCID: PMC6857537  NIHMSID: NIHMS1057414  PMID: 30919347

Abstract

Arrestin proteins were originally characterized as regulators of GPCR desensitization, and that function alone was sufficient to promote extreme interest in their study. It is now appreciated that arrestins also function as mediators of GPCR trafficking and G protein-independent signaling. This latter function places them as prominent players in the emerging field of qualitative signaling, which promises to launch a new area of pharmacology that defines ligands with selectivity/bias toward either G protein-dependent or -independent signaling. To meet the demands of research into arrestin function, methodology has evolved accordingly over the last three decades since the discovery of the arrestin family. Herein we describe state-of-the-art approaches for studying the role of arrestins (β-arrestin1 aka arrestin 2, β-arrestin2 aka arrestin 3) in GPCR function in a primary cell type, cultured airway smooth muscle cells.

Keywords: Arrestin, siRNA, Transfection, Infection, β2-Adrenoceptor, Muscarinic acetylcholine receptor

1. Introduction

Arrestin proteins were originally characterized as regulators of GPCR desensitization, and that function alone was sufficient to promote extreme interest in their study. It is now appreciated that arrestins also function as mediators of GPCR recycling and G protein-independent signaling. This latter function places them as prominent players in the emerging field of qualitative signaling, which promises to launch a new area of pharmacology that defines ligands with selectivity/bias toward either G protein-dependent or G protein-independent signaling. To meet the demands of research into arrestin function, methodology has evolved accordingly over the last three decades since the discovery of the arrestin family.

Early studies of nonvisual arrestins were limited primarily to overexpression of arrestins or dominant-negative arrestin mutants, in cellular expression systems such as COS or HEK293 cells. However, the advent of si/sh RNA methodology, as well as genetic strategies in mice, has provided more powerful approaches capable of greater insight into the role of arrestins in physiologically relevant systems. Herein we describe state-of-the-art approaches for studying the role of arrestins in GPCR function in a primary cell type, the airway smooth muscle (ASM) cells. Generation of cultures of human and murine ASM cells have been previously detailed [1,2].

2. Materials

2.1. cells culture Media

  1. ASM cell culture growth media (maintenance). Complete Ham’s F-12 media with antibiotics: Ham’s F-12 nutrient mix (500 mL) containing 10% FBS (50 mL of invitrogen 10437028), 50,000 units penicillin/50,000 μg streptomycin (P/S) (5 mL of invitrogen 15140122), l-glutamine (5 mL), 12.5 mL of 1 M HEPES, 6 mL of 1 M NaOH, and 850 μL of 1 M CaCl2.

  2. Complete Ham’s F-12 media without antibiotics for ASM cultures subject to transient transfection/virus infection: Ham’s F-12 Nutrient Mix (500 mL) containing 10% FBS, l-glutamine (5 mL), 12.5 mL of 1 M HEPES, 6 mL of 1 M NaOH, and 850 μL of 1 M CaCl2.

  3. Complete DMEM media for virus generation: DMEM high glucose (500 mL) containing 10% FBS and P/S.

  4. DMEM media no P/S for virus generation: DMEM high glucose (500 mL) with 10% FBS only.

  5. Plain DMEM media for virus generation: DMEM high glucose (500 mL) with no additions.

  6. Serum-free media for terminal experiments: Ham’s F-12: Ham’s F-12 Nutrient Mix (500 mL) containing l-glutamine (5 mL), 12.5 mL of 1 M HEPES; 4 mL of 1 M NaOH, P/S, and 794 μL of 1 M CaCl2 insulin/transferrin (IT)-supplemented media are made by adding 5 ng/mL of insulin and 5 ng/mL of transferrin to serum-free Ham’s F-12. Alternatively, serum-free Ham’s F-12 can be supplemented with 0.1% bovine serum albumin (BSA) (30% sterile BSA stock solution).

2.2. ASM Cell Culture

  1. 60 mm and 100 mm cell culture dishes.

  2. ASM cell culture growth media (maintenance) (see Subheading 2.1, item 1).

  3. Phosphate-buffered saline (PBS): 8.1 mM Na2P04,1.47 mM KH2P04,137 mM NaCl, and 2.7 mM KC1, pH 7.4.

  4. Trypsin (0.5% trypsin, 1 mM EDTA).

  5. Micropipette (2–20, 20–200,200–1000 μL).

  6. Serological pipette (2, 5,10, 25 mL).

  7. Phase contrast and/or fluorescence microscope (Nikon Eclipse E800 fluorescence microscope; Plan-Apo 60 × 1.40 NA oil immersion objective; QED Camera software).

  8. Antibiotics for plasmid expression selection (G418 or hygro-mydn, e.g., depending on the plasmid resistance cassette).

  9. Water bath at 37 °C.

2.3. Plasmid Transfection

  1. Fugene HD reagent (Promega E2311) (see Note 1).

  2. DNA plasmids (ARR2-GFP, ARR3-GFP, and ARR2(R169E)- FP) as per [3] (see Note 2).

  3. OptiMEM media.

2.4. siRNA Transfection

  1. Nuclease-free water.

  2. siRNA buffer: 60 mM KC1, 6 mM HEPES, pH 7.5, and 0.2 mM MgCl2 (prepared from 5× stock with nuclease-free water; 1 part 5 × siRNA buffer + 4 parts nuclease-free water; store at 4 °C).

  3. Cell culture media (Ham’s F-12; without additives).

  4. β-arrestinl siRNA (Dharmacon Cat no. L-011971), β-arrestin2 siRNA (Dharmacon Cat no. L-007292) (see Note 3).

  5. DharmaFECTl (Dharmacon).

  6. Cell culture media (Complete Ham’s F-12 media and plain DMEM).

  7. 1.5 mL Eppendorf tubes.

  8. Table top centrifuge.

2.5. Retrovirus Generation

  1. Complete DMEM media.

  2. DMEM media without p/s.

  3. Plain DMEM media.

  4. Fugene HD (see Subheading 2.3, item 1)

  5. 0.45 μm PVDF filter unit.

  6. Complete Ham’s F-12 media with antibiotics.

  7. Complete Ham’s F-12 media without antibiotics.

  8. Polybrence—supplied as 10 mg/mL

  9. G418 sulfate—(500 mg/mL) (see Note 4)

  10. GP2–293 packaging cells.

2.6. Immunoblotting, Second Messenger Assays, mRNA Isolation and Migration Assays

  1. Radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) with protease and phosphatase inhibitor cocktail.

  2. Gel electrophoresis and transfer equipment (Biorad).

  3. Criterion midi 10% polyacrylamide gels (Biorad).

  4. Nitrocellulose blotting membrane.

  5. LiCOR blocking buffer (LiCOR).

  6. Anti-β-arrestinl/2 rabbit mAb (1:1000; Cell Signaling Technology).

  7. LiCOR IRDye secondary anti-rabbit Ab (1:10,000; IiCOR).

  8. Protein Simple Wes apparatus (Protein Simple).

  9. Protein Simple Wes electrophoresis kit (Protein Simple).

  10. Applied Biosystems cAMP-Screen system (Thermo Fisher).

  11. CyQuant NF cell proliferation assay kit (Thermo Fisher).

  12. TRIzol reagent.

  13. mRNA column isolation kit.

  14. Boyden chamber fitted with an 8 μm pore membrane.

3. Methods

3.1. Molecular Strategies: Overexpression of Wild-Type or Dominant-Negative Arrestins

The methodology described here is similar to that previously used by us (see Note 5)

  1. Aspirate media from a 100 mm dish of near-confluent (~3 × 106 cells/dish) ASM cells.

  2. Wish cells twice with 37 °C calcium-free PBS and add 2 mL trypsin; incubate at 37 °C until cells detach (1–2 min).

  3. Resuspend cells in serum-containing media and plate cells in a 60 mm cell culture dish at a density of 12,400 cells/cm2 so as to achieve ~50% confluence the next day.

  4. Twenty-four (24) h after plating cells (now at ~50% confluence), re-feed cells with serum-containing media (3 mL for a 60 mm dish) lacking antibiotics.

  5. Pipette 300 μL of OptiMEM into a 1.5 mL tube

  6. Add 6 μg of DNA (encoding arrestin isoforms or mutants) to the tube and tesuspend gently by pipetting.

  7. Add 18 μL of Fugene HD to the tube; resuspend gently by pipetting.

  8. Incubate for 10 min (see Note 6).

  9. Add dropwise to the cells.

  10. Assess the egression of arrestins by visualization of GFP (see Note 7) or by immunoblot analysis; and/or add selection agents 48 h post-transfection to select for stable expression of arrestins (see Note 8).

  11. Addition of antibiotics is optional at this stage.

  12. Harvest cells and replate them for specific experiments (Fig. 1).

Fig. 1.

Fig. 1

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Expression of arrestin-GFP chimeras in transient and stably transfected human ASM cells, (a) HASM cells were transfected with pcDNA3 vector (Mock), pEGFPN1 (GFF), ARR2-GFP, or ARR3-GFP and analyzed for fluorescence using a Coulter Epics Elite ESP Flow Cytometer. The population of cells exhibiting fluorescence greater than that established in mock-transfected cells (autofluorescence) was sorted and subsequently plated for analysis of arrestin expression, receptor-mediated cAMP production, or agonist-dependent ARR2- GFP localization, (b) Populations of ARR2-GFP- or ARR3-GFP-expressing cells isolated by FACS were plated onto 12-well plates. Four days later cells were harvested, and ARR2-GFP and ARR3-GFP expressions were assessed by immunoblotting using the polyclonal antibodies 178 (specific for arresting) and 182 (specific for arrestin-3). (c) Two separate lines expressing ARR2-GFP were also established by selection with G418, with one line expressing a low level of ARR2-GFP (ARR2-GFP(A); ~three- to fourfold of endogenous arrestin-2 observed in untranslated cells (UNT)) and another line expressing a high level of ARR2-GFP (ARR2-GFP(B); >100-fold of endogenous arrestin-2). Different lengths of autoradiograph exposure account for differences in intensity of bands representing endogenous arrestin-2.

3.2. Molecular Strategies: Arrestin Knockdown Using Transient siRNA Transfection

The strategy described below results in robust knockdown of β-arrestins (see Note 9).

  1. As per Subheading 3.1 methods, passage ASM cells the day prior into 60 mm dishes to achieve ~50% confluence the day of siRNA transfection.

  2. Warm up all buffers and media to 37 °C in a water bath.

  3. Re-feed plated cells with complete Ham’s F-12 media prior to preparing transfection reagents (3 mL per 60 mm dish).

  4. Resuspend lyophilized siRNA to 100 μM in 1 × siRNA buffer. Vortex at the lowest speed or shake lightly by hand to resuspend. Spin the tube down briefly using a tabletop centrifuge at a low speed (100 rcf; ≤1 min).

  5. Prepare three 1.5 mL tubes per siRNA used.

  6. Fill first tube with 100 μL of 1 × siRNA buffer.

  7. Fill second and third tubes with 100 μL plain DMEM cell culture media.

  8. Add 2 μL of 100 μM siRNA solution to the first tube.

  9. Transfer the contents of the first tube to the second tube.

  10. Add 6 μL of DharmaFECT to the third tube.

  11. Incubate for 5 min at RT.

  12. Transfer the contents of the second tube to the third tube.

  13. Incubate for 20 min at RT.

  14. Add the entire contents of the third tube, drop wise, to the cell culture dish.

  15. Assess the effect of protein knockdown 72–96 h post- transfection by immunoblotting.

  16. The procedure can be scaled up or down for other size cell culture dishes (Fig. 2).

Fig. 2.

Fig. 2

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β-arrestin1 and β-arrestin2 expression in human ASM cultures treated with scrambled (SCR), βarr1, or βarr2 siRNA. Values are means ± SEM from nine experiments.

3.3. Molecular Strategies: Stable Arrestin Knockdown, Using a Retrovirus-Mediated Approach

  1. For retrovirus generation, thaw GP2–293 packaging cells and plate into a 10-cm dish containing 10 mL complete DMEM media.

  2. Change cell media to fresh complete DMEM the day after thawing. Depending on the freezing of the cells, there may be many dead/floating cells. Do not discard the plate, just continue to change the media every few days until cells recover, and start to grow. Be careful not to overgrow cells or plate them at a very low density as they will not behave properly.

  3. When cells are confluent, they should be split ~1:4 into new 10 cm dishes. They usually need to be split 2–3 times per week.

  4. When ready to make virus (day 1), split the packaging cells 1:10–12 (assuming a fully or nearly confluent 10 cm dish) into new 10 cm dishes.

  5. On day 2 (transfection day), check to see that cells are ~30% confluent (see Note 10). Change media to DMEM no P/S (10 mL) prior to transfection.

  6. Transfect the packaging cells using Fugene HD.
    • (a)
      Per 10 cm plate/construct—In a sterile Eppendorf tube (in hood), add 5 μg of retroviral vector construct and 5 μg of the helper plasmid (pVSVG) to 485 μL of plain DMEM media (see Note 11).
    • (b)
      Add 15 μL of Fugene HD to the media/DNA mixture. Make sure to add the Fugene HD directly to the media without touching the plastic of the tube!!
    • (c)
      Mix thoroughly and incubate for 15 min at room temperature.
    • (d)
      Add mixture to the 10 cm dish (containing DMEM no P/S media).

  7. Eighteen to twenty-four hours (18–24 h) later (on day 3) change media to 10 mL of complete DMEM. Incubate the culture for another 48 h to allow viral titer to increase.

  8. On day 5, collect the supernatant containing the virus. Filter through a 0.45 μm PVDF filter into a sterile 50 mL conical tube. Virus can be used to infect target cells immediately; if not, freeze filtered virus in the conical tube at −80 °C.

  9. Twenty-four hours (24 h) prior to infection, plate ASM cells at a density of 12,500 cells/cm2 into 10 cm dishes in 10 mL of complete F-12; this should achieve ~50% confluence the next day.

  10. Obtain filtered virus just harvested from packaging cells (or thaw out the frozen virus at 37 °C; see Note 12).

  11. On day of infection, prepare F-12 NO P/S media containing 8 μg/mL polybrene. Aspirate media from the target cells, and add 5 mL of the media/polybrene mixture to each 10 cm dish of target cells.

  12. Add 5 mL of the appropriate, previously prepared virus per dish. Virus amounts may vary, but the logic here is to use one half volume of fresh media (so cells stay fed and happy) plus one half volume of virus which should be of sufficient titer to infect cells. Incubate cells at 37 °C.

  13. After 24 h incubation with virus, remove virus-containing media and replace with fresh complete F-12 media.

  14. The next day (48 h post-virus addition):
    • (a)
      For transient expression, cells can be plated for experiments in complete F-12 media.
    • (b)
      For stable expression, change media to complete F-12 containing 250 μg/mL G418 (250 μL of 500 mg/mL G418 solution per bottle complete F-12). Cells will select/die off over the next 7–14 days. If virus expresses a fluorescent protein, this can be used to monitor selection/infection efficiency. Change media to fresh F-12/ G418 every 3–4 days. Once cells are confluent/no longer dying off, pass into a 15 cm dish to expand and/or plate for experiments.

  15. For infection of lung tissue ex vivo that contains ASM (either isolated airways or precision cut lung slices in culture, from either mice or humans), the tissue infection is performed using lentivirus (see Note 13) as per Morgan et al. [4]. The virus titer, and duration of virus exposure, may need to be empirically determined for each tissue culture and each virus preparation.

3.4. Signaling Functional Experiments in ASM Using Transfected/Infected ASM Cultures

  1. Plating of cells for terminal experiments: After transient transfection or stable line generation, cells are plated at a density of 25,000 cells/cm2 in either 96-, 24-, 12-, or 6-well plates (day 0) (see Note 14).

  2. Cells are re-fed with complete F-12 media on day 3.

  3. On day 6, cells (which should be ~95% confluent) are then washed once with sterile room temperature PBS and re-fed with serum-free media. Twenty-four hours (24 h) later (day 7, if re-red with F-12 media containing 0.1%BSA, or 48 later, if re-red IT media) cells are stimulated for signaling or functional experiments.
    • (a)
      Stimulation of ASM cells for signaling analyses: wash cells with room temperature PBS and re-feed plain Ham’s F12 (see Note 15).
    • (b)
      When ready to stimulate cells with the appropriate agonist, add reagents and place culture dish in either a 37 °C water bath (for experiments of 1 h duration or less) or return to the 37 °C incubator (for experiments of >1 h duration).
    • (c)
      Upon conclusion of stimulation, wash cells in ice-cold PBS twice, then lyse with appropriate lysis buffer for specific experiment as described previously in [5–9].

  4. For analysis of cAMP accumulation, we use the Applied Biosystems cAMP-Screen system [10]; lyse cells with manufacturer provided lysis buffer and perform assay according to manufacturer’s instructions.

  5. For immunoblot analysis, we use standard SDS-PAGE and the Li-Cor Odyssey system as per [5]; lyse cells in RIPA buffer with protease and phosphatase inhibitor cocktail. Perform SDS-PAGE gel electrophoresis and transfer proteins onto nitrocellulose membranes; block with LiCOR blocking buffer (1 h at RT), and incubate membrane with anti-β-arrestinl/2 rabbit mAb (1 h at RT); incubate with secondary IRDye (1 h at RT), and perform detection and quantification as per [5], with discrimination of isoform based on protein size. See Note 16 for protein detection using the ProteinSimple Wes system.

  6. For immunoblot analysis of ASM cells, the current antibody of choice that we use is anti-β-arrestinl/2 rabbit mAb (1:1000, Cell Signaling Technology), with discrimination of isoform based on size.

  7. For immunocytochemistry of ASM cells and immunohisto-chemistry of ASM tissue, no commercially available antibody has been validated (see Note 17).

  8. For analysis of ASM growth, we use the CyQuant assay as per [9] plate cells in a 96-well plate (1 × 104 cells per well) and maintain in complete Ham’s F-12 medium supplemented with 10% FBS. After 24 h, switch to serum-free IT media, and treat with stimuli for 72 h; aspirate media, and add manufacturer-provided assay buffer containing CyQuant dye; incubate for 30 min (RT), and measure fluorescence intensity as per the manufacturer’s instructions.

  9. For analysis of mRNA abundance regulation by quantitative PCR, we lyse the cells in TRIzol and isolate mRNA using a column-based mRNA isolation kit. cDNA generation and quantitative PCR are performed as per [8].

  10. To assess single cell contractility of ASM cells, we use magnetic twisting cytometry (MTC). Dynamic changes in cell stiffness are measured as an indicator of contraction of isolated ASM cells using the MTC technique as per [8]. Briefly Arg-Gly-Asp (RGD)-coated ferrimagnetic microbeads bound to adherent cells are magnetized horizontally and then twisted in a vertically aligned homogeneous magnetic field that was varying sinusoidally in time. This sinusoidal twisting magnetic field caused both a rotation and a pivoting displacement of the bead: such forced bead motions are, in turn, impeded by internal stresses developed by the cell. Lateral bead displacements in response to the resulting oscillatory torque is detected with a spatial resolution of ~5 nanometer (nm), and the ratio of specific torque to bead displacements is computed and expressed as the cell stiffness in units of Pascal (Pa) per nm. For each individual ASM cell, baseline stiffness is measured for the first 60 s, and changes in cell stiffness in response to agonists are measured continuously for the next 240 s. Given this technique requires expensive equipment, software, and considerable expertise, it is typically performed by one of only a handful of labs throughout the world, in a collaborative manner.

  11. For cell migration assays, cells are trypsinized and resuspended in serum-free media supplemented with BSA. Cells (5 × 104) are then placed into the upper wells of a Boyden chamber, fitted with an 8 μm pore membrane. Agonists or vehicle in serum-free media supplemented with BSA are added to the lower chambers. Cells in the Boyden chamber are incubated for 4 h at 37 °C in a 5% C02 incubator. Nonmigrated cells are scraped off. The membrane is then fixed and stained, and the cells are counted as per [6].

Acknowledgment

Studies in the Penn and Pera labs are supported by NIH grants HL58506, HL136209, HL114471, and AI110007 (RBP) and HL140064 (TP).

4 Notes

1.

Other transfection reagents are likely effective. As with any transfection reagents, their efficacy and toxicity should be empirically determined for the specific cell type and protocol employed.

2.

Constructs encoding ARR2-GFP, ARR3-GFP, and ARR2 (R169E)-GFP were generated by polymerase chain reaction amplification of the open reading frames of bovine ARR2, ARR3, and ARR2R169E (all previously cloned into pcDNA3) and cloning in pEGFPNl (CLONTECH, Palo Alto, CA) such that the C-terminal GFP sequence was in frame to generate the intended chimera. Design of dominant-negative arrestin constructs were based on and validated by studies from the Gurevich [11] and Lefkowitz labs [12].

3.

siRNA are ON-TARGETplus SMARTpool oligos (Dharmacon, Lafayette, CO, USA), representing a combination of siRNA empirically established to selectively target human β-arrestinl or β-arrestin2. Single-specific siRNAs targeting β-arrestinl (5’-AAAGCCUUCUGCGCGGAGAAU-3’) or β-arrestin2 (5’AAGGACCGCAAAGUGUUUGUG-3’) are also effective [13].

4.

Dissolve contents of bottle (5 g) in 10 mL dH2O, and filter through a 0.22 μm filter. Aliquot into individual Eppendorf tubes and freeze at −20 °C until needed.

5.

This methodology is similar to that employed in Penn et al. J Biol Chem 2001 [3], the original study assessing the effect of arrestin overexpression on signaling of multiple endogenously expressed Gs-coupled GCPRs (β2-adrenoceptor, EP2/4 receptors, and A2b adenosine receptor) in human ASM. Our early studies on arrestins in ASM, performed prior to the successful application of antisense or si/sh RNA knockdown strategies, employed overexpression of wild-type or dominant- negative arrestin mutants in order to assess which GPCRs in ASM might be susceptible to arrestin-mediated regulation. Although this strategy suffers from the inherent limitation of overexpression strategies (e.g., protein-protein interactions and events occurring solely as a result of supraphysiological expression of a protein), later studies that involved knockdown or knockout of endogenous arrestins would both clarify the physiological role of arrestins and reveal those spurious effects conferred by arrestin overexpression. Our laboratory prefers to use a transfecting scrambled siRNA oligonucleotide to generate control cells. However, comparison of cells transfected with scrambled siRNA oligonucleotides with those (sham) transfected with oligonucleotide vehicle suggest no differences in GPCR signaling or function between these two control lines (Penn, unpublished data).

6.

For Fugene HD it is important not to incubate for too long (max 10 min); transfection efficiency may drop with increasing incubation times.

7.

The strategy of expressing arrestin constructs as GFP chimeras was based on: (1) the ability to easily detect GFP and confirm efficiency of transfection/infection, (2) enabling the option to sort GFP+ cells to further achieve a greater % of cells expressing the recombinant arrestin, and (3) the experimental control and ease of interpretation provided by the use of control cells transfected/infected with GFP alone. Transfection efficiency with this method 48 h after transfection of primary ASM culture using Fugene HD can range from 40 to 75%.

8.

For stable expression, strategies employing retroviral or lentiviral infection (with selection, following the protocol below for stable shRNA expression) are preferable to transient transfection followed by selection. This is due to the greater efficiency of expression/integration that occurs with virus-mediated infection, enabling a greater number of cells to survive selection which in turn allows for a more rapid expansion of cells expressing the construct of interest.

9.

In our hands, this method results in typically ≥75% knockdown of β-arrestinl or β-arrestin2. Dual knockdown can be achieved with similar efficiency. In ASM cells this level of knockdown was shown to increase isoproterenol-induced cAMP response by 50% post- β-arrestin2 knockdown, whereas β-arrestinl knockdown resulted in a 30% decrease of methacholine- induced calcium mobilization [7].

10.

If there are too few cells, they will not produce enough virus. If cells are too dense, they will overgrow before the virus is ready.

11.

Our lab has used multiple viral vectors to generate virus to infect ASM successfully. The choice of vector may depend on the ease of cloning the desired DNA into the vector, the need for co-expressing a fluorescent protein, or the type of selection required. The majority of viral preparations have used the pLNCX2 retroviral vector, which we have employed with great success [4].

12.

This procedure is for unconcentrated virus. The amount of virus added per dish may vary if it is concentrated (or cells other than HASM are used) and should be empirically determined before starting. Aim to end with 10 mL of media/polybrene/virus per 10 cm dish.

13.

Infection of ASM cells with lentivirus, enabling the expression of either recombinant protein or shRNA, follows a similar protocol with the preparation of lentivirus differing as per [4]. As with retrovirus, each lentivirus preparation requires empirical assessment of the amount of virus that produces optimal expression/knockdown with minimal toxicity.

14.

Typically, optimal (highest) expression of arrestin recombinant proteins expressed via transient transfection occurs 48–72 h post-transfection, whereas the highest level of arrestin isoform knockdown occurs at 96 h post-transient transfection. Pretreatments, serum starvation, and cell cycle arrest steps for various functional assays should be timed accordingly, if optimal expression/knockdown is desired.

15.

For select acute signaling experiments, such as assessing short duration treatment effects on intracellular cAMP accumulation or phosphoprotein induction, cells can be refed PBS (with Ca2+ and Mg2+). Solubility of stimuli in media/buffer is a factor to consider. If pre-treating cells with reagents, return cells to 37 °C incubator.

16.

Protein Simple Wes is a fully automated capillary electrophoresis apparatus used for immunodetection of (phospho-)proteins [14]. After stimulation cells are washed with ice-cold PBS and lysed in RIPA buffer, and lysates are loaded on manufacturer-provided plates and run in the Wes apparatus according to manufacturer’s instructions. Data are analyzed using manufacturer-provided software as per [10].

17.

Few published studies exist in which endogenous arrestins are detected using either immunocytochemistry or immunohistochemistry for any cell type or tissue. Overwhelmingly, the consensus among labs known for studying arrestins is that immunocytochemical and immunohistochemical detection of endogenous arrestins requires affinity-purified arrestin antibodies produced in academic labs.

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