Figure 3.

HECTD1 promotes BIRC6 ubiquitination and degradation during EGFR activation

(A) Coomassie blue staining of proteins co-precipitated with an anti-Myc or anti-BIRC6 antibody. Bottom: the number of unique peptides of HECTD1 identified by mass spectrometric analysis. (B) Hs578T cell lysates were immunoprecipitated with an HECTD1 antibody and subjected to immunoblotting analysis. (C) Hs578T cells expressing Flag-tagged HECTD1 were treated with or without MG132 for 8 h and then immunoblotted with the indicated antibodies. (D) Hs578T cells expressing Flag-tagged HECTD1 were treated with CHX (100 μg/mL) for the indicated times, followed by immunoblotting as indicated. (E) Scramble or HECTD1-knockdown Hs578T cells were treated with CHX (100 μg/mL) for the indicated times and then immunoblotted with the indicated antibodies. (F) After being transiently transfected with the indicated plasmids, 293T cells were immunoprecipitated with an anti-Myc antibody and then subjected to immunoblotting analysis. Cells were treated with 10 μM MG132 for 6 h before being lysed. (G) Scramble or HECTD1-knockdown Hs578T cells were immunoprecipitated with an anti-BIRC6 antibody and subjected to ubiquitination analysis. (H) After being serum starved overnight, scramble or HECTD1-knockdown Hs578T cells were treated with EGF for 30 min, followed by immunoblotting as indicated. (I) After being treated with or without EGF, Hs578T cells were immunoprecipitated with an anti-HECTD1 antibody and subjected to immunoblotting analysis.