C3G and p38α MAPK interact with FA proteins. (A) Representative immunoprecipitation assays (IP), using the indicated antibodies, performed with protein extracts from K562 cells cultured on 10 μg/ml fibronectin for 24 h. Expression of p33, p46 and p68 phospho-paxillin (p-Pax) and paxillin isoforms, p38α, p87C3G, CrkL, Bcr-Abl, p130Cas, Abi1 and FAK was detected by immunoblotting with specific antibodies. GammaBind G Sepharose beads (B) with either buffer or whole cell lysate (lysate) were used as negative controls. (B) Confocal microscopy of K562 cells adhered for 24 h to slides covered with 10 μg/ml fibronectin and labeled with the indicated antibodies. Nuclei were labeled with DAPI. Control cells (CT) were incubated with DAPI plus the secondary antibodies. C3G-1008 (rabbit polyclonal) was used with Pax (mouse monoclonal) and p-FAK (goat polyclonal). C3G-G4 (mouse monoclonal) was used with p-Pax (rabbit polyclonal). C3G colocalizes with p-Pax and p-FAK in a punctuated pattern (white arrows). DIC: Differential interference contrast microscopy. Pax: paxillin. A488: Alexa Fluor 488. The bars represent 5 μm.