Fig. 5. PKC is involved in phosphorylation of AFAP-110 on Ser277. (A,B) Kinase inhibitors were added to GFP-AFAP-110 transfected COS-7 cells before PMA treatment for 1 hour. Staurosporine (STS) to inhibit Ser/Thr kinases and PP2 for Src kinase (A), and Safingol (Saf) and rottlerin (Rot) to selectively inhibit certain PKC isoforms as well as other Ser/Thr kinases (B). Cell lysates were separated by SDS-PAGE, and western blotted with antibodies against anti-pSer277 ((P) AFAP, upper panel), or anti-GFP (lower panel) to control for equal loading. (C) PKC or the dominant-active myristoylated (myr-) form of PKC were co-expressed with GFP-AFAP-110 in COS-7 cells and cells were stimulated with 100 nM PMA for 1 hour as indicated. Control cells were left untransfected and also subjected to PMA treatment as indicated. Cells were directly lysed in sample buffer and subjected to SDS-PAGE and western blot analysis using anti-pSer277 ((P) AFAP; top panel), anti-GFP ( GFP; middle panel) or anti-PKC ( PKC; bottom panel). All blots are labeled with molecular mass markers on the left. (D) Similarly, cells expressing GFP-AFAP-110 were pre-treated with GF109203X (GF), followed by stimulation with 100 nM PMA for 1 hour as indicated. Cells were directly lysed in sample buffer and subjected to SDS-PAGE and western blot analysis using anti-pSer277 ((P) AFAP, top panel) or anti-GFP ( GFP; bottom panel).