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

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Down-regulation of IRS4 by CK1γ2 is mediated by lysosomes in cells.(A) 293T and osteosarcoma cells were co-transfected with HA-tagged wild type (WT) or kinase-depleted(KD) CK1γ2 plasmids and Flag-IRS4 for 48 h and analyzed by Western blotting. (B) The indicated cells were transfected with the indicated shRNAs and then were analyzed by Western blotting. (C) 293T cells were co-transfected with HA-CK1γ2 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. (D) 293T cells were co-transfected with Flag-IRS4 and HA-CK1γ2 and HA-Ub(K48WT) or HA-Ub(K63WT). Cell lysates were subjected to immunoprecipitation with anti-Flag-agarose and then analyzed by Western blotting.