Figure 6.

Proteasome activity and clathrin-mediated endocytosis modulate HGF/SF and EGF-induced Hrs phosphorylation. (A) K44A cells were cultured for 48 h with (+) or without (−) tetracycline and then stimulated with HGF/SF for the times indicated. The cells were then lysed and proteins immunoprecipitated with anti-Hrs antibody. Immunoprecipitated proteins were analyzed by immunoblotting with PY20 antibody (top) or anti-Hrs (bottom). (B and C) HeLa cells were preincubated for 2 h with (+) or without (−) lactacystin and then stimulated with HGF/SF or EGF for the times indicated. Analysis of Hrs phosphorylation was performed as in A. (D) Intensity of phospho-Hrs bands (top) and total Hrs bands (bottom) from C were quantitated using NIH Image 1.62 software and a ratio of phospho-Hrs:Hrs was calculated for each time point. Phospho-Hrs:Hrs after 20-min stimulation was expressed as a percentage of phospho-Hrs:Hrs after 8-min stimulation. Each data point represents mean ± SEM of three experiments. *p < 0.05.