Figure 3
Indispensable role of SOS1 in the ErbB2/HER2 signaling cascade. (a) Efficacy of shRNAs targeting SOS1. MKN45 cells were transduced with control (sh_Luc) or with SOS1 shRNAs (sh_SOS1#1 and #2) and cultured in the presence of 3 μM doxycycline. Twenty-four hours after treatment, total RNA was prepared and analyzed by real-time RT-PCR. Values were normalized to that of control vector-transduced cells (n = 3). (b) SOS1-depletion-mediated dephosphorylation of HER2. Non-depleted and SOS1-depleted MKN45 cells were treated as in (a). Forty-eight hours after treatment, cell lysates were processed for immunoblotting. (c) Growth curves of MKN45 cells transduced with control (sh_Luc) or with SOS1 shRNAs (sh_SOS1#1 and #2) in the presence of 3 μM doxycycline (n = 5). (d) Efficacy of shRNAs targeting HER2. MKN45 cells were transduced with control (sh_Luc) or with HER2 shRNAs (sh_HER2#1 and #2) and cultured in the presence of 3 μM doxycycline. Twenty-four hours after treatment, total RNA was prepared and analyzed by real-time RT-PCR. Values were normalized to that of control vector-transduced cells (n = 3). (e) HER2-depletion-mediated dephosphorylation of HER2. Non-depleted and HER2-depleted MKN45 cells were treated as in (d). Forty-eight hours after treatment, cell lysates were processed for immunoblotting. (f) Growth curves of MKN45 cells transduced with control (sh_Luc) or with HER2 shRNAs (sh_HER2#1 and #2) in the presence of 3 μM doxycycline (n = 5). (g,h) Immunoprecipitation assay showed SOS1-HER2 complex in MKN45 cells. Data are mean ± SEM values. *P < 0.05, **P < 0.01, ***P < 0.001, by two-tailed Student’s t-test.
