Figure 4
PSAP down-modulation decreased FAK activity and prevented β 1A -integrin clustering and proper assembly of focal adhesion complex. (A) PSAP down-modulation reduced phosphorylation of FAK and paxillin. Cells were incubated in suspension with gentle rotation for 45 min and then plated onto FN- or LN-coated dishes for 45 or 90 min. Whole cell lysates were extracted and equal amount of proteins were used for immunoprecipitation with anti-FAK or-paxillin antibody and immunoblotting with phospho-specific antibodies against Tyr-397, -576, -861, -925 of FAK or Tyr-118 of paxillin. (B) Effect of PSAP down-modulation on β1A-integrin clustering and focal adhesion complex assembly. Cells were plated onto FN- or LN-coated slides for 2 h, fixed and permeabilized. Immunofluorescence staining was performed with primary antibodies against integrin β1A, FAK pY397 and paxillin pY118 followed by Cy3 (red) or FITC (green)-conjugated secondary antibodies. F-actin was stained by Oregon Green 488-phalloidin (green). All images were taken by a Leica DM RA2 fluorescence microscope. Consistent data were obtained from three independent experiments.