Fig. 6. EGF signaling disrupts SID–mSin3A interaction. (A) CHO cells were transiently transfected with FLAG-tagged full-length TIEG2 along with vErbB or caMEK1 as indicated. Anti-FLAG immunoprecipitations were performed, and TIEG2 and mSin3A were detected by western blotting. Note that TIEG2 strongly binds endogenous mSin3A (lane 1) while vErbB or caMEK1 co-expression significantly reduces the binding of mSin3A by TIEG2 (lanes 2 and 3, respectively). The MEK1 inhibtor PD089059 reverses the vErbB-mediated reduction of mSin3A binding by TIEG2 (lane 4). Anti-TIEG2 western blot analysis shows comparable expression of TIEG2. (B) CHO cells were transfected with GAL4 NTD, NTD deletion mutants (see Figure 5) or GAL4 DBD alone. Anti-GAL4 immunoprecipitations were performed and mSin3A co-immunoprecipitation was examined by western blotting. Note that mSin3A co-immunoprecipitates with GAL4 NTD (lane 2) and deletion constructs D2 and D3 (lanes 6 and 8, respectively), but not with D1 (lane 4). CaMEK1 co-expression greatly reduces the binding of mSin3A to NTD (lane 3) and D3 (lane 9), but not to D2 (lane 7). Controls show that GAL4 DBD alone (lane 1) does not co-immunoprecipitate with mSin3A. (C) CHO cells were transfected with GAL4 NTD or D3 constructs carrying the M5 mutations. Note that mSin3A co-immunoprecipitation with NTD M5 (lanes 3 and 4) and D3 M5 (lanes 1 and 2) is not changed by co-expression of caMEK1. Western blot analysis shows that all GAL4 constructs exhibit comparable expression levels.