FIGURE 6.
Grb2 knockdown affects downstream effectors activation. A, TS cells were transduced with lentiviral particles for different sequences (A, B, C, and control (Ctrl)) of doxycycline-inducible (DOXY) Grb2 shRNA (shGrb2). After 96 h of induction, Grb2 knockdown and its effects on the downstream effectors were tested by Western blot with the indicated antibodies. B, densitometry analysis of blots from four independent experiments as described in A was performed to quantify the variation of expression of Grb2 and of downstream proteins phosphorylation. Histograms in the left panel indicate the relative expression level of Grb2 as percentage of the expression levels detected in non-transduced cells. The difference in relative expression between shGrb2A or -B and control was evaluated using Student's t test. Grb2 expression was significantly reduced by both shRNA (*, p < 0.01). The right panel shows the relative phosphorylation levels of the indicated proteins as percentages of the phosphorylation levels detected in non-transduced cells. The difference in relative phosphorylation between both shGrb2 and control was evaluated using Student's t test. SHC and SHP2 phosphorylation was significantly reduced by shRNA (*, p < 0.005; **, p < 0.001). In contrast, ERK phosphorylation was not significantly affected by shRNA. C, TS TTA (doxycycline-inducible) cells were infected with Grb2 shRNA (A or B) alone or together with shRNA-resistant Grb2 constructs (Grb2WTINT3/4 or Grb2Y160FINT3/4). Cells were cultured for 96 h with (+) or without (−) 1 μg/ml doxycycline (DOXY). Cell lysates were analyzed by Western blot with the indicate antibodies.