Overexpression of the Nedd4-1-binding-defective mutant ACK1Y650A and the ubiquitination-defective mutant ACK1Δ89 inhibits EGF-induced degradation of EGFR. (A) ACK1 RNAi or luciferase RNAi (control RNAi) was transfected into A549 cells for 60 h followed by 12 h serum starvation. The cells were stimulated with EGF (50 ng/ml) for 0, 30, and 60 min. The amounts of EGFR and ACK1 were detected by immunoblotting with anti-EGFR (1005) and anti-ACK (A11). (B to E) pcDNA3 (vector control), pcDNA3-Myc-ACK1, pcDNA3-Myc-ACK1Y650A, or pcDNA3-Myc-ACK1Δ89 was transfected into HEK293 cells for 36 h followed by 12 h serum starvation. The cells were stimulated with EGF (50 ng/ml) for 0, 5, 30, and 60 min (B and C) or 0, 30, and 90 min (D and E). EGFR was immunoprecipitated with anti-EGFR (Mab528) and immunoblotted with anti-EGFR (1005). The amount of tubulin (B) or actin (D) was used to indicate lysate loading. In panels C and E, the amount of EGFR determined by immunoblotting was quantified from two independent experiments by FUJIFILM Multi Gauge V3.0.