FIGURE 4.

GRB7 knock down in trastuzumab and lapatinib resistant, HER-2 positive breast cancer cell lines reduced HER-1 tyrosine phosphorylation. A, Cell lysates were first immunoprecipitated with anti-phospho-tyrosine antibody (Cell Signaling Technology, #9419) followed by blotting and staining with anti-HER-1 (Cell Signaling Technology, #4267), HER-2 (Cell Signaling Technology, #2242), and GRB7 antibodies. B, Cell lysates were immunoprecipitated with anti-HER-1 antibody (Cell Signaling Technology, #8083) blotted with anti-HER-1 (Cell Signaling Technology, #4267) and anti-phospho-tyrosine antibodies (Cell Signaling Technology, #8954). C, Cell lysates were blotted with anti-phospho-HER-1 antibody (Cell Signaling Technology, #3777). EV: empty vector; KD: GRB7 knock down. D, HCC1954 cells were infected with three different shRNA targeting vectors: Clone ID: GRB7 KD = TRCN0000061385; GRB7 KD2 = TRCN0000061384; GRB7 KD3 = TRCN0000061386, (GE Healthcare, Dharmacon). Cell lysates were blotted with anti-phospho-HER-1 antibody (Cell Signaling Technology, #2220), anti-total HER-1, anti-GRB7, and anti-GAPDH antibodies