Figure 2

Asymmetric kinase dimer formation is dispensable for ERBB3 activation by EGFR kinase. (A) Indicated EGFRvIII and ERBB3 constructs were transfected into HEK293 cells. After 36 hours, stimulation with 50 ng/ml of heregulin for 5 minutes was performed. Cells were then lysed and subjected to immunoprecipitation using anti-ERBB3 antibody. Immunoblotting was performed on ERBB3 immunoprecipitates with indicated antibodies (top 2 panels). Whole cell lysates (input) were subjected to western blotting with indicated antibodies (lower 3 panels). (B) Schematic representation of receptor interactions corresponding to the experiments shown in Figure 2A (lanes 3, 4, 7 and 8). (C) Indicated EGFR and ERBB3 mutants were transfected into HEK293 cells followed by serum starvation and heregulin (50 ng/ml) stimulation for 5 minutes. Immunoblotting on ERBB3 immunoprecipitates was performed with indicated antibodies (upper 2 panels). Immunoblotting of whole cell lysates (input) was performed with indicated antibodies (lower 2 panels). (D) Wild-type EGFR was co-transfected with either wild-type or C-lobe mutant (V945R) ERBB3 into HEK293 cells. After 36 hours, cells were serum starved for 12 hours followed by stimulation with EGF (25 ng/ml) or heregulin (50 ng/ml) for 5 minutes. Cells were then lysed and subjected to ERBB3 immunoprecipitation. SDS-PAGE and immunoblotting on ERBB3 immunoprecipitates was performed with indicated antibodies (upper 2 panels). Western blotting on whole cell lysates (input) was performed with indicated antibodies (lower 2 panels). (E) EGFR-L858R was co-transfected with either wild-type or C-lobe mutant (V945R) ERBB3 into HEK293 cells. After 36 hours, cells were serum starved for 12 hours followed by stimulation with EGF (25 ng/ml) or heregulin (50 ng/ml) for 5 minutes. Cells were then lysed and subjected to ERBB3 immunoprecipitation. SDS-PAGE and immunoblotting on ERBB3 immunoprecipitates was performed with indicated antibodies (upper 2 panels). Western blotting on whole cell lysates (input) was performed with anti-EGFR antibody.