FIG. 2.

Inhibition of GSK3 in G₀/G₁-arrested cells leads to a disappearance of specific phospho forms of p130. (A) Inhibition of GSK3 in serum-starved cells results in dephosphorylation of the endogenous p130 in T98G glioblastoma cells. Panel A shows a Western blot (WB) of p130 in extracts prepared from T98G cells that were arrested in G₀ by serum deprivation (lanes 2 to 8) and left untreated (lanes 2 and 3) or treated for 3 h with 25 mM lithium acetate (lanes 4 and 5) or 25 μM GSK3 inhibitor II (lanes 7 and 8). Cell extracts were resolved by SDS-PAGE (6% polyacrylamide gel) prior to blotting onto the nitrocellulose paper. To compare the migration of GSK3 inhibitor-treated and dephosphorylated p130, samples in lanes 2, 5, and 8 were treated with λ-phosphatase (λ-Pptase) prior to loading. A sample prepared from cycling T98G cells (10% FBS, lane 1) is shown to emphasize the difference in gel migration of specific phospho forms of p130 (forms 1a, 1b, 2, and 3, shown to the side). (B) Enhanced GSK3 phosphorylation of p130 from lithium-treated cells compared to intact cells. T98G cells were arrested in G₀ by serum deprivation and left untreated (lanes 1 to 4) or treated for 3 h with 25 mM lithium acetate (lanes 5 to 8) prior to lysis and immunoprecipitationwith anti-p130 C-20 antibodies. Control samples were immunoprecipitated in the presence of the C-20 blocking peptide (BP) to ensure the specificity of the reactions (odd lanes). Samples were resolved by SDS-PAGE (7.5% polyacrylamide gel) and transferred to nitrocellulose. The upper panel shows an autoradiogram of p130 (³³P-p130) and the PhosphorImager quantification of the p130 signal. Western blot panels of p130 and GSK3B show that equal amounts of these proteins were present in each reaction. (C) Serum restimulation of BJ/hTert fibroblasts results in transient dephosphorylation of p130, coinciding with inactivation of GSK3. BJ/hTert cells were synchronized in G₀ and then treated with 20% serum for the indicated time. Cell extracts were resolved by SDS-PAGE (6% polyacrylamide gel for p130 and 10% polyacrylamide gel for GSK3 analysis, respectively) prior to blotting onto nitrocellulose paper. Western blots show the changes in phosphorylation of p130 occurring during the exit of the cells from the G₀-arrested state and the kinetics of GSK3 inactivation, monitored by phosphorylation of the serine 9 residue (pS9) of GSK3B. The membrane was stripped and reprobed with anti-GSK3B antibodies to ensure equal loading of the samples (Gsk-3β). (D) FACS analysis of DNA content of BJ/hTert cells from the experiment shown on panel C. (E) Serum restimulation results in a transient decrease of G₀/G₁ phosphorylation of p130 in T98G cells, while treatment with lithium leads to a sustained loss of G₀/G₁ phosphorylation. The left panels show a Western blot of p130 in T98G cells that were serum starved for 72 h to induce the G₀/G₁ state (lane 3) and treated with 25 mM lithium acetate (lanes 1 and 2) or 20% fetal calf serum (lanes 4 to 7) for the indicated time. The right panels show a Western blot of the HA-tagged recombinant human p130 stably expressed in NIH 3T3 cells that were serum starved and treated as described above. Cell extracts were resolved by SDS-PAGE (6% polyacrylamide gels) prior to blotting. Membranes were reprobed with antivinculin antibodies to demonstrate equal loading of the samples (WB: Vinc).