Wang et al. 10.1073/pnas.0601682103. |
Supporting Figure 8
Supporting Figure 9
Supporting Methods
Fig. 8. Characterization of the polyclonal anti-Su48 antibody Ab1. (Upper) The polyclonal anti-Su48 antibody Ab1 can bind to the C-terminal portion of Su48. U2OS cells were transfected with various Su48 plasmid constructs [lane 1, GFP; lane 2, GFP-Su48(1-45); lane 3, GFP-Su48(1-374); lane 4, GFP-Su48(1-184); lane 5, GFP-Su48(1-291); lane 6, GFP-Su48(1-315) or GFP-Su48]. The cell lysates were prepared and subjected to Western blot analyses using Ab1. (Lower) Expression of GFP fusion proteins in U2OS cells. To confirm the expression of the GFP fusion proteins, cell lysates were subjected to Western blot analysis using a monoclonal anti-GFP antibody.
Fig. 9. Characterization of the monoclonal anti-Su48 antibodies 24F1 and 8H9. (A) Fluorescence microscopy image of U2OS cells transfected with GFP-Su48 after immunostaining using the anti-Su48 antibody 24F1. (B) Immunoprecipitation of FLAG-tagged Su48 using the anti-Su48 antibodies 24F1 or 8H9. 293T cells were transfected with FLAG-Su48. The cell lysate was subjected to immunoprecipitation using either anti-FLAG antibody M2, 24F1, or 8H9. The proteins bound to the antibodies were separated by SDS/PAGE and analyzed by Western blot using anti-FLAG antibody. (C) g-Tubulin does not associate with FLAG-Su48 in coimmunoprecipitation analysis. 293T cells were transfected with FLAG-Su48. Immunoprecipitation was performed using either anti-FLAG antibody M2, 24F1, or 8H9. The proteins bound to the antibodies were separated by SDS/PAGE and analyzed by Western blotting using anti-g-tubulin antibody (lanes 2, 3, 4, and 5). In lane 1, an aliquot of the cell lysate was loaded.
Materials and Methods
Cell Cycle Synchronization.
Cell cycle synchronization was achieved by a thymidine/nocodazole double-block protocol. To obtain cells in the G2/M stage, cells were cultured in medium containing 5 mM thymidine for 12 h, washed with fresh medium, released from the block for 4 h, and then incubated in medium containing 1 mM nocodazole for 8 h. An aliquot of these cells were confirmed to be in the G2/M stage by DNA staining and FACS analysis. Cells were collected, washed, and released from the second block by culturing in fresh normal medium. Cells were examined after 6 h (G1 phase) or 14 h (S phase). To destabilize the microtubule network, cells were incubated with 1 mM nocodazole for 4 h. Disruption of the microtubule network was confirmed by immunostaining with the anti-a-tubulin (B512) antibody.Immunostaining.
Cells grown on acid-washed coverglass were fixed first in buffer A (3% paraformaldehyde in PBS) for 5 min at room temperature and then in cold methanol for 5 min. The cells were then incubated in blocking buffer (20 mM Hepes, pH 7.5, 50 mM NaCl, 3 mM MgCl2 with 0.1% Triton X-100, and 2% BSA) for 15 min and stained with the primary antibody in the blocking buffer for 30 min. After three washes in PBS, the cells were incubated with the secondary antibody in blocking buffer. Finally, the cells were mounted in VectaShield medium containing DAPI (Vector Laboratories) and analyzed by deconvolution fluorescence microscopy (Leica DMIRBE inverted microscope and OPENLAB 3.1.4 software).Production of Antibodies.
The recombinant GST-Su48(156-407) fusion protein was used to generate polyclonal antibody in rabbit. The antisera were first partially purified by ammonia sulfate precipitation and cleared off anti-GST reactive species by passing through a GST column. The remaining antiserum was affinity-purified using a GST-Su48(156-407) column. The anti-serum (Ab1) was confirmed to detect Su48 by Western blot analysis.To generate anti Su48 monoclonal antibodies, BALB/c mice were immunized s.c. with purified His-Su48 fusion protein, and hybridoma cell clones were obtained by conventional hybridoma technology followed by ELISA screening. The hybridoma clones producing the antibody reactive with His-Su48 were selected and subcloned. The hybridoma 24F1 produces an IgG1a that detected FLAG-Su48 in transfected U2OS cells by immunostaining. The monoclonal antibodies were purified from hybridoma culture medium with G protein.
Antibody Microinjection.
HeLa cells were grown on coverslips until 50% confluency and injected with antibodies using glass capillary needles pulled on a micropipette puller. The mouse monoclonal anti-Su48 antibody 24F1 or an isotype-matched control antibody was dialyzed in PBS and injected at a concentration of 5 mg/ml. Twenty-four hours after injection, cells were fixed first in PBS with 3% paraformaldehyde and then in methanol, followed by staining with the Cy3-conjugated anti-mouse antibody. The coverglass was mounted in VectaShield medium containing DAPI. At least 200 cells were injected and counted for each sample. The experiment was repeated twice.RT-PCR Analysis.
To examine Su48 expression in cell lines, cDNA samples were generated by reverse transcription using total RNA isolated from HeLa, U2OS, PANC-1, HBL100, and 293T cells. Total RNA was extracted using TRIzol reagent (Invitrogen). The gene expression pattern in mouse tissues was studied with cDNA samples prepared from mouse tissues or the mouse embryonic day 11 cDNA library were used for PCR (BD Biosciences Clontech).The following primers were used for PCR of the human Su48 gene:
Forward: 5'-ggaattcaatgcaacagaaggcttttgagg-3';
Reverse: 5'-ggaattcgatgatgttcacaatggctgttgg-3'.
Primers used for PCR of the mouse Su48 homolog (GenBank accession no. AF543761) are:
Forward: 5'-gggagcaacaagtccggaatttc-3';
Reverse: 5'-gtgggccctcaaggatgacaagcttc-3'.
The primer set for the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene was used in control reaction:
Forward: 5'-tgaaagtcggagtcaacggatttggt; Reverese: 5'-catgtgggccatgaggtccaccac.
PCR was programmed for 30 cycles of reaction at the melting temperature of 94°C (45 seconds); annealing temperature of 65°C (45 seconds); and extension temperature of 72°C (2.5 min).
In vitro
Protein Binding Assay. To make the fusion construct GST-Su48(156-407), the cDNA of the C-terminal fragment of Su48 was subcloned to the pGEX-5X plasmids (Amersham Pharmacia). GST-Su48(156-407) was expressed in bacteria and isolated with glutathione Sepharose beads according to the manufacturer’s instructions. The isolated protein was examined by SDS/PAGE. The full-length Su48 protein was expressed and isotope-labeled using the TnT in vitro translation system (Promega). Glutathione Sepharose beads loaded with »5 mg of GST or GST fusion proteins were incubated with 10 ml of in vitro translation system products in 300 ml of binding buffer (50 mM Tris, pH 7.5/100 mM NaCl/0.2% Nonidet P-40/1% BSA/1 mM DTT). After 1 h of incubation at room temperature, the beads were precipitated and washed three times with the binding buffer. The protein bound to the beads was resolved by 10% SDS/PAGE and detected by PhosphorImager (Molecular Devices). A sample of the in vitro translation product (5 ml) was loaded on the gel to show the size and the amount of the input protein.Immunoprecipitation and Western Blotting.
293T cells were transfected with a combination of plasmids using the FuGene 6 reagent (Roche Applied Science, Indianapolis). Twenty-four hours after transfection, cell lysates were prepared in lysis buffer (120 mM NaCl/25 mM Tris·HCl, pH 7.4/0.5% Nonidet P-40) supplemented with the protease inhibitor mixture (Roche Applied Science). After clearing the cell lysate by centrifugation, the supernatant was incubated first with 5 mg of antibodies at 4°C for 1 h and then with 20 ml of protein G/protein A agarose bead suspension (Invitrogen). The proteins bound to the agarose beads were separated by SDS/PAGE and analyzed by Western blotting. The horseradish–peroxidase-conjugated anti-mouse IgG k light chain antibody was used as the secondary antibody.Yeast two-hybrid analysis.
The yeast strain HF7C was transformed with a combination of "bait" and "prey" plasmid constructs. The plasmid pGBT-Su48 carries the fusion between the Gal4 DNA-binding domain and full-length Su48; pGBT is the parental plasmid that contains the coding region for the Gal4 DNA-binding domain only. pGAD-Su48 encodes the fusion between the Gal4 transcription activation domain (AD) and Su48; pGAD contains the Gal4-AD only. The transformed yeast clones were tested for activation of the Gal4-regulated His-3 reporter, by virtue of the ability to grow on the synthetic medium devoid of histidine.