Fig. 3. Characterization of the anti-pSer277 antibody as a potential phospho-specific AFAP-110 antibody. (A) Specificity of the newly generated phospho-specific AFAP-110 antibody was determined by dot-blot assay as described in Materials and Methods. NP, non-phosphorylated peptides; P, phosphorylated peptides. (B) Western blot analysis with the newly generated phospho-specific AFAP-110 antibody anti-pSer277. Samples were prepared as described in Fig. 2A,C, where a gel shift of the AFAP-110 protein band was observed in response to PMA treatment in western blot analysis. Here the western blot was probed with anti-pSer277 phospho-specific AFAP-110 antibody ((P) AFAP, upper panel) or with an antibody against total AFAP-110 (AFAP, lower panel) to control for equal expression of AFAP-110. (C) Time course of PMA-induced immunoreactivity with anti-pSer277 antibodies. GFP-AFAP-110 transfected COS-7 cells were treated with 100 nM PMA for the indicated time, prior to lysis in sample buffer, separation by SDS-PAGE and subsequent western blot analysis with anti-pSer277 ((P) AFAP, upper panel). After stripping the membrane it was re-probed with a GFP antibody (lower panel), to control for equal loading. (D) Anti-pSer277 antibodies are immunoreactive with AFAP-110 in cells treated with PMA/PDBu. Anti-pSer277 antibody ((P) AFAP, upper panel) recognizes PMA/PDBu-induced phosphorylation of overexpressed avian and human AFAP-110 as well as of endogenous human and rat AFAP-110 in western blot analysis. Overexpression of AFAP-110 was performed in COS-7 cells that had been lysed directly after PMA treatment. Endogenous AFAP-110 was immunoprecipitated after PMA treatment, as indicated, and SDS-PAGE and western blot analysis was performed. Membranes were probed with anti-pSer277 antibodies ((P) AFAP, upper panel) and with a anti-AFAP antibody (lower panel), to control for equal loading. All western blots are labeled with molecular mass markers on the left.