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. Author manuscript; available in PMC: 2012 Jun 1.
Published in final edited form as: Cell Signal. 2011 Jan 22;23(6):1030–1040. doi: 10.1016/j.cellsig.2011.01.018

Figure 1. BCAR3-mediated anti-estrogen resistance and Rac activation are independent of BCAR3-p130Cas complex formation.

Figure 1

(A) A clonal MCF-7 cell line stably transfected with HA-tagged BCAR3 (II-6 cells) or polyclonal MCF-7 cell populations stably transduced with either a control lentivirus or wildtype or R743A HA-tagged BCAR3-expressing lentivirus were immunoprecipitated with HA antibody and probed with p130Cas antibody (left panel). Whole cell lysates (WCL) were probed with HA antibody to demonstrate expression of the exogenous BCAR3 and with p130Cas antibody to assess phosphorylation of endogenous p130Cas as determined by a shift in p130Cas gel mobility (right panel). (B) MCF-7 cells stably transfected with HA-tagged BCAR3 were transiently transfected with wildtype or L791D p130Cas, followed by immunoprecipitation with BCAR3 antibody and probing with an HA antibody. WCL from these two cell types as well as MCF-7 cells transiently transfected with wildtype p130Cas were probed with HA antibody to examine the expression and electrophoretic mobility of exogenous BCAR3 and p130Cas. (C) Following treatment with 100nM fulvestrant (ICI 182,780), parental MCF-7 cells or polyclonal populations stably transduced with empty, wildtype BCAR3, R743A BCAR3, NSP3 or NSP3/BCAR3 GEF-expressing lentivirus were assessed for cell growth. Experiments were performed three times. Asterisks indicate significant differences in cell growth (p < 0.05). (D) The PAGE migration of p130Cas was examined in WCL of either wildtype MCF-7 cells or polyclonal MCF-7 cells stably transduced with control, wildtype or R743A BCAR3 or wildtype or chimeric NSP3/BCAR3 GEF lentivirus. Expression of the NSP family members was confirmed by probing with HA antibody. (E) GST-PAK1 pulldown assay to detect GTP-bound Rac in MCF-7 cells transiently transfected with empty vector, HA-BCAR3(WT), HA-BCAR3(R743A) or HA-BCAR3(ΔGEF). WCL were probed for total Rac and HA. The pulldown assay was performed in triplicate.