Fig. 2. Ubiquitination of overexpressed Hrs is constitutive and independent of tyrosine phosphorylation. (A) HeLa cells were transfected with GFP-Hrs, GFP-Y329F, GFP-Y334F, GFPY329/334F or GFP-UIM, starved for 16 hours in serum-free medium and then stimulated 22 hours post-transfection for 8 minutes with EGF (100 ng/ml) or left unstimulated. Lysates were prepared in the presence or absence of 10 mM NEM and subjected to immunoprecipitation with anti-GFP antibody. Ubiquitination was assessed by immunoblotting with anti-ubiquitin antibody. Molecular mass markers are indicated on the left. (B) HeLa cells were transfected with GFP-Hrs, GFP-Y329F, GFP-Y334F, GFPY329/334F, GFP-LSAA, GFP-UIM or mock transfected, and treated as in A. Lysates were prepared according to the `boiling SDS-lysis' method (see Materials and Methods) and subjected to immunoprecipitation with anti-GFP antibody.