Fig. 5. Insulin-triggered Rho activation requires annexin A2. (A) BHK-IR cells were transfected with either control (mock) or annexin A2 siRNA or with the RhoQ63L expression construct. Cells were treated with 100 nM insulin as indicated, fixed and probed for Rho GTP loading state with GST-RBD as described in the Materials and Methods. Bound GST-RBD was detected by staining with anti-GST-antibody. F-actin was visualized with TRITC-phalloidin. Note the insulin-induced increase in GST-RBD binding that is not seen in annexin-A2-downregulated cells. Scale bars: 10 µm. (B) Bound GST-RBD was quantified using the MetaMorph software (mean values ± s.e.m.). ^*P<0.05.