Fig. 1. BCAR3 regulates p130Cas phosphorylation in human breast cancer cell lines.
Panel A: Three epithelial ERα-positive and three ERα-negative mesenchymal breast cancer cell lines were assessed for BCAR3 and p130Cas expression by Western analysis. Panel B: p130Cas was immunoprecipitated from the indicated breast cancer cell lines, treated with lambda phosphatase in the presence or absence of EDTA, and examined by Western analysis. Panel C: Upper panel: MCF-7 cells were transfected with vector alone or the same vector driving expression of NSP1, NSP2 (BCAR3) or NSP3, followed by Western blotting with an anti-p130Cas antibody. Lower panel: MCF-7 cells were transfected with a construct driving expression of hemagglutinin epitope-tagged p130Cas (HA-Cas) in combination with vectors driving expression of HA-tagged NSP1, NSP2 or NSP3, as indicated, followed by Western blotting with an anti-HA antibody. The vector panel is shown at shorter exposure and the NSP1 panel at longer exposure than the NSP2 and NSP3 panel due to varying levels of HA-Cas expression in their respective whole cell lysates. Panel D: Three MDA-231 cell lines were isolated in which the Tet-repressor inhibits expression of an shRNA targeting BCAR3 expression in the absence of doxycycline. The three lines, as well as a control MDA-231 line containing a lentiviral shRNA construct targeting GFP, were treated for four days in the presence or absence of doxycycline, followed by Western analysis for expression of BCAR3 and p130Cas.