Fig. 1. Tyrosine phosphorylation of Hrs in response to EGF. (A) An antibody directed against an Hrs peptide containing phospho-Y334 recognizes Hrs specifically in response to EGF stimulation. HeLa cells were starved for 16 hours in serum-free medium and either not stimulated or stimulated for 8 minutes with EGF (100 ng/ml). Lysates were analysed by immunoblotting with anti-PY334-Hrs antibody. Molecular mass markers are indicated. (B,C) EGF-dependent tyrosine phosphorylation of Hrs is dependent on the presence of either Y329 or Y334 as well as on an intact UIM domain. (B) HeLa cells were transfected with GFP-Hrs, GFP Y329F, GFP-Y334F, GFP-Y329/334F, GFP-UIM, GFP-LSAA or mock-transfected, starved 16 hours in serum-free medium and then stimulated 22 hours post-transfection with EGF (100 ng/ml). Lysates were prepared and subjected for immunoprecipitation with anti-GFP antibody. Phosphorylation was assessed by immunoblotting with PY20 antibody. (C) Lysates were prepared as described in b and analysed by immunoblotting with anti-GFP and anti-PY334-Hrs antibodies respectively. The arrowhead indicates GFP-PY334-Hrs, the arrow indicates endogenous PY334-Hrs. (D) PY334-Hrs is enriched in the cytosol. HeLa cells were starved for 16 hours in serum-free medium and stimulated for 8 minutes with EGF (100 ng/ml) or left unstimulated. Membrane and cytosol fractions were prepared as described in the Materials and Methods. The relative distribution of tyrosine phosphorylated Hrs was assessed by analysing equal proportions of membrane and cytosolic fractions by immunoblotting with anti-Hrs and anti-PY334-Hrs antibodies. (E) Membrane and cytosol fractions were prepared from cells stimulated for 8 minutes with EGF, and the specific enrichment of Hrs phosphorylated at Y334 was analysed by loading four times more membrane fraction than cytosol fraction on SDS-PAGE followed by immunoblotting with anti-Hrs and anti-PY334-Hrs antibodies as in D.

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