Figure 3.

Down-regulation of IRS4 by the E3 ligase CHIP is mediated through lysosomes in cells. (A) 293T cells were co-transfected with Myc-CHIP and Flag-IRS4 for 48 h, and then lysed and immunoprecipitated using anti-Flag-agarose or anti-MYC-agarose followed by Western blotting. (B) Cell lysates from 293T cells were immunoprecipitated with anti-CHIP or anti-IRS4 and were subsequently analyzed with anti-IRS4 or anti-CHIP antibody. (C) The indicated cells were infected with the indicated plasmids and then were analyzed by Western blotting. (D) U2OS and 293T cells were infected with the indicated shRNAs and then were analyzed by Western blotting. (E) U2OS cells stably infected with control shRNA or CHIP shRNA were treated with 40 μg/mL CHX for the indicated periods and then analyzed by Western blotting.(F) 293T cells were co-transfected with Myc-CHIP and Flag-IRS4 for 48 h and then treated with Bortezomib or Bafilomycin A for 6 h. Cell lysates were then analyzed by Western blotting.