Figure 5

PKCα positively regulates Rac activity in C1861.9 cells. A) C8161.9 or M14 cells were placed in suspension (S), plated on vitronectin (VN) for 45 minutes, or confluent cells were serum starved and then stimulated with 40 uM IGF (IGF) for 5 minutes. The level of GTP-bound Rac (RacGTP) was measured using GST-PBD pull-down assays (PBD IP) and immunoblotting for Rac activity. Total levels of Rac were monitored by immunoblotting of whole cell extracts (Rac Blot). B) C8161.9 cells uninfected (control) or infected with wild type (WT Src) or dominant interfering Src mutant (SrcKM) virus were treated and plated as in A) and analyzed for Rac activation (PBD IP). C) C8161.9 cells were pretreated with DMSO, TPA for 16 hours, or bisindolylmaleimide (Bis). Or cells were transfected with nothing (C), scrambled (Scr), PKCα (iPKα), or PKCδ (iPKδ) siRNA and the total levels of PKCα, PKCδ, and PKCε were monitored by immunoblotting. Total levels of protein were monitored by immunoblotting for tubulin. D) C8161.9 cells were treated with DMSO or bisindolylmaleimide, or transfected with scrambled (Scr), PKCα (iPKα), or PKCδ (iPKδ) siRNA and their ability to activate Rac (PBD IP) in suspension (S) or after plating on vitronectin (VN) was assessed as in A). E) Cells were treated as in D) and PAK activation was measured by immunoblotting of whole cell lysates with anti-phospho-PAK antibody that recognizes the activated form of PAK (P-PAK Blot). Total levels of PAK in the cell lysates were monitored by immunoblotting with anti-PAK antibodies (PAK Blot).