FIGURE 3.
Phosphorylation of p27-Ser10 decreases in HIPK2-depleted cells. A, endogenous proteins derived from H1299-CV and H1299i cells were immunoprecipitated (IP) with anti-p27kip1 Ab. Immunocomplexes were subjected to SDS-PAGE, and nearly equal amounts of immunoprecipitated proteins were loaded and analyzed by WB using Abs specific for p27kip1 and its phosphorylated form at serine 10. B, left panel, TCEs used for the immunoprecipitations were analyzed by WB. γ-Tubulin expression shows equal loading of samples. Right panel, densitometric analysis of phosphorylated p27-Ser10 normalized for total p27kip1 protein level (already normalized to γ-tubulin). Means ± S.D. (lines) of five independent experiments, including the one shown in the left panel, are reported. *, p < 0.01. C, H1299-CV and H1299i cells were treated with DMSO or MG132 for 16 h. The p27kip1 protein level and p27-Ser10 phosphorylation were analyzed by WB. γ-Tubulin expression shows equal loading of samples. One representative of three independent experiments is shown.