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. 2018 Jun 7;8(13):3643–3653. doi: 10.7150/thno.26021

Figure 4.

Figure 4

The E3 ligase CHIP is involved in the phosphorylation-dependent degradation of IRS4 by CK1γ2. (A) 293T cells were co-transfected with Flag-tagged IRS4-WT or -S859A with Myc-CHIP for 48 h. Cell lysates were subjected to immunoprecipitation with anti-MYC-agarose and then analyzed by Western blotting. Cells were treated with 200 nM Bafilomycin A for 6 h before harvesting. (B) 293T cells were co-transfected with Flag-IRS4-WT or -S859A with Myc-CHIP and HA-Ub. Cell lysates were subjected to immunoprecipitation with anti-Flag-agarose and then analyzed by Western blotting. (C) 293T cells were co-transfected Myc-CHIP with Flag-tagged IRS4-WT, -S856, or -859A for 48 h and then analyzed by Western blotting. (D) Cell lysates from 293T cells transfected with Myc-CHIP, Flag-IRS4 and HA-CK1γ2 were subjected to immunoprecipitation with anti-MYC-agarose. WCL and immunoprecipitates were then analyzed by Western blotting. Cells were treated with 200 nM Bafilomycin A for 6 h before harvesting. (E) 293T cells were transfected with Myc-CHIP and Flag-IRS4. 24 h after transfection, the cells were treated with 200 nM Bafilomycin A with or without 25 μM CK1 inhibitor CKI-7 for 8 h prior to cell collection. Cell lysates were subjected to immunoprecipitation with anti-MYC-agarose and then analyzed by western blotting.