FIG. 2.

Effect of βPix depletion by siRNA expression on growth factor-induced ROS generation. (A) Two different cell lines (Caco-2 cells and HEK293T cells) were transfected with either pSUPER-βPix, encoding a βPix-specific siRNA, or pSUPER-Vav2, encoding a Vav2-specific siRNA. Control cells were transfected with only the vector control (pSUPER). After being cultured for 48 h, the cells were deprived of serum for 12 h. After EGF treatment for 10 min, the generation of H₂O₂ was then monitored by confocal microscopic analysis of DCF fluorescence. Data are means ± SE of values from five independent experiments. (B) Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to βPix and Vav2 (Babraham Co., Cambridge, United Kingdom); the filter was reprobed with antibodies to β-actin. (C) HEK293T cells were transfected with pSUPER (control) or pSUPER-βPix. After being cultured for 48 h, the cells were deprived of serum for 12 h. After EGF treatment for 10 min, cell lysates were then prepared with PBS containing 1% Triton X-100 and were incubated for 3 h with a GST fusion protein of PAK-RBD conjugated to glutathione-Sepharose 4B beads. The beads were then washed with ice-cold cell lysis buffer, and bound proteins were subjected to immunoblot analysis with antibodies to Rac1 (upper panel). Cell lysates were also directly subjected to immunoblot analysis with antibodies to Rac (second panel), βPix (third panel), and β-actin (bottom panel).