Figure 2
From: Involvement of βPIX in angiotensin II-induced migration of vascular smooth muscle cells
βPIX mediates Ang II-induced migration of VSMCs. (A) Inhibitory effect of βPIX depletion on VSMC migration. Cells were transfected with βPIX-specific siRNA (100 nM) or scrambled siRNA. After 48 h, the wound migration assay was conducted. Representative images are shown (left) and relative migration data are expressed as mean ± SD (center). Expression of βPIX was confirmed by Western blotting (right). (B) βPIX-dependent Rac1 activation and targeting by Ang II. Cells were stimulated with 100 nM Ang II for the indicated times (left) or with increasing concentrations for 10 min (center). Cell lysates were immunoprecipitated with anti-βPIX antibody followed by incubation with 2 µg purified soluble GST-PBD. Bound GST-PBD representing active Rac1 in the immunoprecipitates was detected by Western blotting with anti-GST antibody (top). Immunopreciptated βPIX and bound Rac1 were confirmed by immunoblotting with anti-βPIX (bottom) and Rac1 (middle) antibodies, respectively. To analyze βPIX-dependent Rac1 targeting by Ang II, cells were transfected with the indicated siRNAs for 48 h (right). Membrane fractions from siRNA-treated cells were immunoblotted with anti-Rac1 (top) or βPIX (bottom) antibody.
