C3G modulates the expression and activation of FA proteins. (A) Western blot analysis of whole cell lysates (50 μg of protein) from K562 cells stably transfected with pLTR2C3G or the empty pLTR2 vector grown in suspension for 24 h. Paxillin (Pax), Cbl, CrkL, FAK, p130Cas and integrin α5 (Int α5) expression and paxillin and CrkL phosphorylation were detected with specific antibodies (B) Comparative analysis of the expression of Int α5, FAK, Pax and p130Cas in the above K562 clones grown with 10 μg/ml fibronectin (FN) for 24 h or in suspension (Susp). β-actin and β-tubulin (ß-Tub) levels were used as loading controls. (C) Western blot analysis of whole cell lysates from K562 cells stably transfected with shC3G (clone 1) or shCT lentivirus, either grown with fibronectin (FN) or in suspension (Susp) for 24 h. FAK, Cbl, paxillin, p130Cas and CrkL expression and paxillin, p130Cas and CrkL phosphorylation were detected. Relative ratios between the levels of these proteins and ß-tubulin are shown. (D) Paxillin expression is decreased in C3G silenced cells. Confocal microscopy of control (shCT) and shC3G-1 K562 cells adhered to fibronectin and labeled with anti-paxillin-Cy3 and anti-phalloidin antibodies. Nuclei were labeled with DAPI. Control cells (CT) were incubated with DAPI plus the secondary antibodies. The bars represent 10 μm (rows 1 and 3) and 7.5 μm (rows 2 and 4). (E) Immunoprecipitation assays (IP) of K562 cell lysates expressing, either shC3G or shCT, with the indicated antibodies followed by Western blot analysis of CrkL, Paxillin, p130Cas and Bcr expression. GammaBind G Sepharose beads (B) with either buffer or whole cell lysate (lysate) were used as negative controls. L: total cell lysate, Pax: paxillin, pY: phospho-tyrosine.