Fig. 9. Cdc42 is required for BMP2-dependent PAK1 and PAK4 phosphorylation. (A) C2C12 cells transfected with the indicated expression vectors were serum-starved for 16 hours and stimulated with 3 nM BMP2 for 40 minutes. Cell lysates were analyzed with the indicated phospho-specific antibodies, and membranes reprobed with anti-PAK1, anti-PAK4 and anti-Cdc42 antibodies. Graph indicates PAK1-P and PAK4-P levels of BMP2-treated cells relative to their respective untreated controls. Mean ± s.e.m. of three independent experiments (*P<0.05, unpaired t-test). (B) PAK activity is required for BMP2 to phosphorylate the LIMK1 associated to the BMPRII tail. Immunoblot analysis of C2C12 cells overexpressing His-tagged BMPRII under a tetracyclin (Tet-off)-regulated promoter (left panel). Cells overexpressing His-tagged BMPRII were transfected with the expression constructs indicated, serum-starved for 16 hours and stimulated with 3 nM BMP2 for 40 minutes. Cell extracts were subjected to immunoblotting to detect the total and phosphorylated LIMK1 after Ni²⁺-NTA-agarose purification (right panel).