Figure 10.

The ACK1-Uba domain is required for EGFR degradation. The COS7 cells were transfected with Myc-tagged ACK1 and the ACK1 Uba domain deletion mutant ACK1ΔUba. The controls were transfected with the vector. The cells were serum-starved for 12 h and subsequently stimulated with EGF (100 ng/ml) for 0.5, 1, 2, and 3 h. For the negative controls of EGFR degradation, the cells were treated with the 26S proteasome inhibitor MG-132 (10 μM) for 30 min before the EGF stimulation. The EGFR degradation was assessed by immunoblotting the whole-cell lysates with an anti-EGFR antibody and quantified by Kodak EDAS290 system (Eastmann Kodak, Rochester, NY). Two independent experiments were performed, and data from both experiments were consistent. (A) The immunoblot of the cell lysates with anti-EGFR antibody (top panel). The lysate loading is shown by immunoblotting with anti-β-actin (bottom panel). (B) The plot of the quantification of the immunoblot from the average of two independent experiments by Kodak EDAS290 system.