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. 2017 Aug 9;8(51):88599–88612. doi: 10.18632/oncotarget.20100

Figure 4. β-TrCP2 overexpression induced K48-linked ZNF281 ubiquitination while knockdown of β-TrCP2 had the opposite effect.

Figure 4

(A1) Endogenous proteins in total lysates of HCT116 cells were subjected to IP with indicated antibody followed by immunoblotting with anti-β-TrCP2 or anti-ZNF281 antibody. A rabbit IgG was included as an IP negative control. (A2) HCT116 cells were transfected with ZNF281 and β-TrCP2 constructs. The cell lysates were subjected to immunoprecipitation with indicated antibody and then analyzed by Western blotting. (B) HCT116 cells were transfected with the indicated amounts of expression vectors. Then cell lysates of indicated cells were subjected to immunoprecipitation with anti-ZNF281 antibody and were analyzed by Western blotting with anti-ubiquitin antibody, K48-specific ubiquitin antibody, and K63-specific ubiquitin antibody. (C) HEK293T cells were infected with lentiviral β-TrCP1 shRNA, or nontargeting shRNA, or lentiviral β-TrCP2 shRNA. The knockdown efficiency was analyzed by human β-TrCP1- or β-TrCP2-specific qRT-PCR or Western blotting. (D) Lysates of the β-TrCP1- or β-TrCP2 knockdown or control cells were subjected to immunoprecipitation with anti-ZNF281 and analyzed by Western blotting with antiubiquitin antibody.