Depletion of endogenous clathrin and caveolin-1 inhibits ligand-dependent internalization of the IR-A.
A, gene knockdown for clathrin and caveolin-1 in R−/IR-A cells was achieved by siRNA. Level of clathrin and caveolin-1 in vehicle (Veh), control oligo (Control), and siRNA-treated (siClath or siCav) cells were assessed by immunoblot using anti-clathrin- and anti-caveolin-1-specific polyclonal antibodies. Total protein load was assessed using anti-β-actin polyclonal antibodies. Blots are representative of three independent experiments. Insulin and IGF-II-mediated internalization of the IR-A in R-IR-A cells depleted of endogenous clathrin (B) or caveolin-1 (C) was assessed by ELISA 72 h post-transfection. The data are presented as mean ± S.D. of three independent experiments. Statistical significance was determined using two-way ANOVA with Bonferroni's multiple comparison test. **, p < 0.01; *, p < 0.05 (siRNAs versus oligo-control-treated cells).