Figure 8.

β-PIX is localized to the plasma membrane in ARF6-depleted cells. (A–C) Cells stably expressing the HA-AT₁R were transfected with Flag-β-PIX (A) and Flag-β-PIX and a control scrambled siRNA (60 nM; B) or with Flag-β-PIX and ARF6 siRNA (#1, 60 nM; C). Cells were stimulated with Ang II for 0 or 30 min, fixed, and stained for the distribution of actin, using phalloidin coupled to Alexa-488, and β-PIX, using a specific anti-Flag polyclonal antibody and a secondary anti-rabbit antibody coupled to Alexa-568. This figure is representative of more than 20 cells observed in four independent experiments. (D) HEK 293 cells stably expressing the HA-AT₁R were transfected with a control scrambled siRNA (60 nM) or with our ARF6 siRNA (#1, 60 nM). Cells were left untreated (□) or stimulated with Ang II (30 min; ■). Membranes fractions were prepared, and amounts of associated β-PIX protein were assessed by Western blotting using an anti-β-PIX antibody. Results represent the mean ± SEM of four independent experiments. *p < 0.05, **p < 0.01.