Figure 1.

HGF stimulation increases phosphorylation of Gab1 and enrichment of pGab1 within CCPs. (A) RPE cells were stimulated with either 5 ng/mL EGF or 50 ng/mL HGF for 5 minutes. Whole-cell lysates were prepared, resolved by immunoblotting, and probed with anti-phospho-EGFR (Y1068), anti-phospho-Gab1 (Y627) and anti-phospho-Akt (S473) antibodies. (B-C) RPE cells stably expressing Tag-RFP-T fused to clathrin light chain (RFP-CLC) were stimulated with 50 ng/mL HGF and immunostained for pGab1. Shown in (B) are representative micrographs obtained by TIRF-M (scale 5 μm, arrowheads indicate pGab1-positive CCPs, selected manually). Images obtained by TIRF-M were subjected to automated detection of clathrin structures followed by quantification of pGab1 and RFP-CLC in each detected object, as in.⁵ Shown in (C) is the mean pGab1 fluorescence intensity detected within clathrin structures in the presence and absence of HGF compared to randomized control (> 35 cells per condition from 3 independent experiments).