Fig. 6.

cIAP1 reduces EGFR stability, but promotes its gene transcription. a EGFR and cIAP1 interaction was tested in BT549 cells using PLA assay. b BT549 cells stably expressing Myc/Flag-tagged EGFR were serum-starved and stimulated with 20 ng/ml EGF for the indicated times. Cells were lysed and EGFR was immunoprecipitated with anti-Flag antibody. Western blot was performed to evaluate the interaction of ectopic EGFR with cIAP1 and c-Cbl. c BT549 and MCF10A cells stably expressing Myc/Flag-tagged EGFR were serum-starved, pre-treated with 100 (BT549) or 50 (MCF10A) µg/ml cycloheximide for 30 min and stimulated with 20 ng/ml EGF for the indicated times. Levels of ectopic EGFR were detected with anti-Myc antibody. d Ectopic EGFR was immunoprecipitated as described in Fig. 6b from BT549 cells transfected with control and cIAP1-specific siRNAs. Western blot shows total levels of c-Cbl and the amount of c-Cbl interacting with EGFR. e BT549 and f MCF10A cell stably expressing ectopic Myc/Flag-tagged EGFR were further transduced with lentiviral particles to overexpress c-Cbl or GFP as a control. Cells were silenced for cIAP1, serum-starved, and stimulated with 20 ng/ml EGF as described above, and analyzed by western blot to evaluate the levels of ectopic EGFR by using a Myc-tagged-specific antibody. g BT549 (left panel) and MCF10A (right panel) were silenced for cIAP1 and analyzed by real-time PCR to quantify the levels of EGFR expression fold relative to GAPDH. BT549: unstimulated siCtr vs. sicIAP1 *P = 0.0134, EGF 3 h siCtr vs. sicIAP1 *P = 0.0270; n = 3. MCF10A: unstimulated siCtr vs. sicIAP1 ***P = 0.0004, EGF 3 h siCtr vs. sicIAP1 **P = 0.0183; n = 4; two-tailed paired t-test