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. 2004 Oct;24(20):8970–8980. doi: 10.1128/MCB.24.20.8970-8980.2004

FIG. 3.

FIG. 3.

In vivo phosphorylation of p130 detected by phospho-specific antibodies to GSK3 sites in p130. (A) Characterization of the phospho-specific antibodies raised against pS982/pT986. Whole-cell extract prepared from G0-arrested T98G cells was run in a preparative single-well gel (7.5% acrylamide), transferred to nitrocellulose, and incubated with the C-20 anti-p130 antibody (lane 1) or ppRb2 phospho-specific anti-p130 antibodies (both at 200 ng/ml) in a multi-slot screening cassette. Phospho-specific antibodies were used directly (lanes 2 and 6) or after preincubation with the indicated blocking peptides. Blocking peptides were used at following concentrations: nonphosphorylated peptide (non-P) at 20, 200, and 2,000 ng/ml and the double-phosphorylated phosphopeptide (pS/pT) at 2, 20, and 200 ng/ml. WB, Western blot. (B) Phospho-specific ppRb2 antibodies do not cross-react with p130 mutants with inactivated GSK3 phosphorylation sites. The top panel shows HA-tagged p130, 1,3,5/A, and 2,4,6/A mutants stably expressed in NIH 3T3 cells, immunoprecipitated (IP) with anti-HA antibodies, resolved by SDS-PAGE (6% polyacrylamide gel), and blotted with ppRb2 antibodies. Parental NIH 3T3 cells wereused as a control (lane 1). The cells were arrested by serum deprivation for 48 h prior to the experiment. The blot was reprobed with anti-HA antibody to show comparable amount of proteins in the immunoprecipitates (bottom panel). (C) Inhibition of GSK3 in G0-arrested cells leads to a decreased recognition of p130 by ppRb2. T98G cells were arrested in G0 by serum deprivation and left untreated (lanes 1 and 2) or treated with lithium acetate (25 mM, lane 3), GSK3 inhibitor II (25 μM, lane 4), SB-216763 (30 μM, lane 5), or SB-415286 (100 μM, lane 6) for 3 h. Untreated extracts in lane 1 were incubated with λ-phosphatase (λ-Pptase) before loading. The upper panel shows the ppRb2 Western blot of whole-cell extracts resolved by SDS-PAGE (6% polyacrylamide gel). The bottom panel shows the same membrane after stripping and reprobing with C-20 anti-p130 antibody.