FIG. 1.

Effect of βPix overexpression on EGF-induced ROS generation in Caco-2 or HEK293T cells. (A) Caco-2 or HEK293T cells were transfected with an empty vector (pFLAG-CMV) or a vector encoding FLAG-tagged βPix or Myc-tagged Vav1. After incubation with EGF for 10 min, the generation of H₂O₂ was monitored by confocal microscopic analysis of DCF fluorescence (see Materials and Methods). The mean fluorescence intensity of DCF in experiments was measured and expressed relative to that of unstimulated cells transfected with empty vector. Data are means ± standard errors (SE) of values from five independent experiments. (B) HEK293T cells were transfected with empty vector (pFLAG-CMV) or with a vector encoding FLAG-tagged βPix. Cells were stimulated with EGF for 10 min and then stained with DCF-DA. The fluorescence was analyzed in 10,000 cells each with FACSCalibur (Becton Dickinson), with excitation at 488 nm and emission at 530 nm. (C) Cell lysates from each sample from panels A and B were prepared and subjected to immunoblot analysis (WB) with antibodies to FLAG (for recombinant βPix) or Myc (for recombinant Vav1); the filter was reprobed with antibodies to β-actin.