(A) Serum-starved HEK293 cells, transfected with control or Gankyrin siRNA, were treated with EGF for the indicated times. The lysates of these cells were subjected to immunoprecipitation with indicated antibodies. The relative amount of the coimmunoprecipitated RhoA normalized to the immunoprecipitated RhoGDI, in 0 minutes, is designated as 1.0, and the numbers under the first row indicate the relative amount of RhoA for other time points. (B) As described in Figure 2A, NIH3T3 cells were induced with 4-OHT for the indicated times. The lysates of these cells were subjected to immunoprecipitation with anti-RhoGDI1 antibody and immunoblotting with indicated antibodies. (C) SDS-PAGE and autoradiography of NIH3T3-ER:RasG12V-control shRNA and Gankyrin shRNA cells, which were serum starved overnight and treated for 0–48 hours with 4-OHT. Extracts immunoprecipitated with anti-ROCK2 were incubated with a substrate of a fusion protein of GST-MYPT1 (amino acids 654–880). Phosphorylation of MYPT1 was then detected by autoradiography. (D) RhoA WT or RhoA Q63L mutant were overexpressed with Flag-Gankyrin or vector through infection with retroviral vectors in NIH3T3 cells. Extracts immunoprecipitated with anti-ROCK2 were incubated with a substrate of a fusion protein of GST-MYPT1. We then detected the phosphorylation of MYPT1 with the indicated phospho-specific antibodies. (E) MEF-Pten WT and MEF-Pten knockout cells stably expressed Gankyrin through infection with a retroviral vector encoding human Gankyrin. After that, the cells were serum starved overnight and then treated with EGF for the indicated times. The lysates of these cells were analyzed by immunoblot using indicated antibodies.