Figure 6. POH1 depletion and ErbB2 receptor turnover.

A, HeLa cells were treated±POH1 siRNA for 48 hours before incubation with 10 µg/ml cycloheximide. Cells were lysed and analysed by immunoblotting with ErbB2 29D8 and Ab20 antibodies, which recognize intracellular and extracellular epitopes of ErbB2 respectively, EGFR, and tubulin antibodies. B, quantitation shows that both EGFR (by antibody 1005) and ErbB2 (by antibodies Ab20 and 29D8) are turned over more rapidly in POH1 knock-down cells (data averaged from 3 experiments). C. HeLa cells were treated with four On Target Plus oligos (POH1) or with oligofectamine alone for 72 hours before lysis with hot lysis buffer. A higher molecular weight ErbB2 “smear” was observed in all 4 knock-down samples. D The high molecular weight smear associated with ErbB2 immuno-reactivity is sensitive to treatment with a deubiquitinase (USP2). HeLa cells were treated with POH1 siRNA or oligofectamine for 48 hours before lysis in the presence of NEM. ErbB2 was immunoprecipitated and treated in vitro with USP2 catalytic domain (100 nM, 8 hours, 37°C). Samples were analyzed by immunoblotting with ErbB2 antibodies targeting extracellular (Ab20) and intracellular (29D8) domains. Note that the smear detected with Ab20 is lost upon USP2 treatment whilst detection with the intracellular domain antibody increases. As a control for USP2 DUB-activity, EGFR was immunoprecipitated from EGF-stimulated (5 min) HeLa cells and treated in vitro with USP2 catalytic domain before SDS-PAGE and western blotting with anti-Ubiquitin.