Figure 6.

erbB-2 gene targeting in stable HeLa cell clones. (A) ErbB-2 Western blot. The indicated E2C-KRAB- and E2C-VP64-expressing clones were maintained in the presence or absence of 2 μg/ml Dox for 4 days. Protein extracts from these cells were subjected to Western blotting with the ErbB-2 specific antiserum 21N. Lane C, HeLa/tet-off extract. (B) Northern blot. Total RNA extracted from the indicated cell lines maintained in the absence of Dox for 4 days was subjected to Northern blotting with an erbB-2 specific probe. The membrane was stripped and reprobed with a glyceraldehyde-3-phosphate (GAPDH)-specific probe as a control. (C) Epidermal growth factor (EGF)-induced tyrosine phosphorylation of ErbB-2. The indicated cell lines were maintained in the absence of Dox for 4 days, serum starved overnight, and either induced with 100 ng/ml EGF for 10 min at room temperature or left untreated. ErbB-2 was immunoprecipitated (IP) from protein extracts with antiserum 21N and analyzed by Western blotting (WB) with mAb PY20. (D and E) Flow cytometric analysis of ErbB-2 and ErbB-1 expression. Cells were maintained for 4 days in the absence of Dox, stained with mAbs FSP77 or EGFR1 in combination with phycoerythrin-labeled secondary antibody, and analyzed for their fluorescence in a FACScan (Becton Dickinson). Dotted lines, control staining (primary antibody omitted) of HeLa/tet-off cells; dashed lines, specific stainings of HeLa/tet-off cells; solid lines, specific stainings of, respectively, Dox-deprived KRAB clone 27 and VP64 clone 18.