Figure 2.

PPM1G dephosphorylates p27 at T198. A. The catalytically inactive mutant of PPM1G (D496N) fails to reduce p27pT198. 293T cells were transfected with Flag-p27 and increasing amounts of Flag-PPM1G, WT or DN mutant. PPM1G, p27 and p27pT198 levels were determined by Western blot. B. MG132 has no effect on PPM1G-mediated p27 dephosphorylation. Experiments were carried out similarly as described in A. Cells were treated with MG132 (10 µM) for 8 h. C. PPM1G dephosphorylates p27 in vitro. Phosphorylated p27 was immunoprecipitated from 293T cells, and mixed with PPM1G purified from E. coli as GST fusion protein in the phosphatase reaction buffer. Levels of p27pT198, p27pS10, p27 and PPM1G were determined by Western blot.