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Fig. 1. Interaction of Hrs with clathrin. (A) Schematic representation of the mouse Hrs protein, with its different domains indicated. Proline- and proline/glutamine-rich regions are also indicated. VHS, domain conserved in Vps27, Hrs and STAM; FYVE, domain conserved in Fab1, YOTB, Vac1 and EEA1; UIM, uibiquitin-interacting motif; CC, coiled-coil domain; CB, clathrin-box motif (the residues are indicated in single-letter code). (B) Recombinant GST-clathrin-TD was immobilised on glutathione-Sepharose beads and incubated with cell lysate from HeLa cells transiently transfected with the Hrs constructs indicated. The beads were pelleted and analysed by SDS-PAGE and immunoblotting with an anti-myc antibody. (C) Recombinant GST or GST-clathrin-TD were immobilised on glutathione-Sepharose beads and incubated with cell lysate from HeLa cells transiently transfected with Hrs. The cells were serum-starved for 4 hours and stimulated or not with 50 ng/ml EGF for 15 minutes before lysis. The beads were pelleted and analysed by SDS-PAGE and immunoblotted with an anti-myc antibody. The lysate was also subjected to immunoprecipitation with the anti-myc antibody and immunoblotted with an anti-phosphorylated-tyrosine antibody to detect phosphorylated Hrs. (D) HEp-2 cells were transfected with Hrs-specific siRNA or a scrambled-sequence RNA and subjected to western blotting to detect the efficiency of Hrs knockdown (upper panel). Tubulin is shown as a loading control (lower panel). CB, clathrin-box; IB, immunoblot, IP, immunoprecipitation; TD, terminal domain; PS, Ponceau S-staining.
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