Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Figure 1. Ang II promotes the activation of overexpressed Rac1. (A) HEK293 cells stably expressing HA-AT₁R were transiently transfected with Rac1-myc, and treated with Ang II for the indicated times. Cells were lysed and activated Rac1 captured using GST-PAK(CRIB) coupled to glutathione-Sepharose 4B beads in a GST pull down assay. Levels of activated Rac1 were subsequently detected by Western blotting using anti-myc antibodies. (B) Data are expressed as mean ± S. E. M. (fold/basal) of four independent experiments. ** P < 0.01.
• Supplemental Figure 2 - AT₁R-stimulation promotes Rac1 and ARF6 complex formation. HEK293 cells stably expressing HA-AT₁R were stimulated with Ang II (1μM) for the indicated times and solubilized. Endogenous Rac1 was immunoprecipitated using the polyclonal anti-Rac1 antibody and endogenous interacting ARF6 proteins were then detected by Western blot analysis, using a specific anti-ARF6 antibody. The Rac1 and ARF6 content of the total cell lysate (4% of the initial input) were also assessed. Data are representative of four independent experiments.
• Supplemental Figure 3 - Figure 3. Depletion of ARF1 does not affect basal or Ang II stimulated membrane ruffling in HEK 293 cells. (A) Cells stably expressing the HA-AT₁R were transfected with ARF1 siRNA (25 nM). Cells were stimulated with Ang II for the indicated times, fixed and stained for the distribution of actin using Phalloidin coupled to Alexa-488. The scale bar represents 10 μm. This figure is representative of more than 30 cells observed in four independent experiments. (B) Western blots depicting the expression of endogenous ARF1 in non-transfected, control scrambled siRNA (25 nM), and ARF1 directed siRNA (25 nM) stably expressing HA-AT1R cells. (C) ARF1 knockdown is presented as mean ± S. E. M. of five independent experiments. *** P < 0.001.