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. 2018 Mar 1;9(10):879–889. doi: 10.1007/s13238-018-0510-2

Figure 3.

Figure 3

CKI synergizes with β-TRCP to mediate ZNRF3 degradation. (A–D) IB analysis of IP and WCL derived from HEK293 (A and C) or HeLa (B and D) cells transfected with indicated constructs, cell lysates were treated with or without phosphatase (PPase) for 30 min (A) or cells were treated with MG132 for 12 h (C) before harvesting. (E) IB analysis of WCL derived from HeLa cells infected with lentivirus for control (sh-Scr) or multiple independent shRNAs against CKIδ (sh-CKIδ). Infected cells were selected with 1 μg/mL puromycin for 72 h to eliminate non-infected cells before harvesting. (F) IB analysis of WCL derived from HeLa cells treated with different concentrations of CKI inhibitor D4476 overnight before harvesting. (G) IB analysis of ubiquitin-products and WCL derived from HEK293 cells transfected with indicated constructs treated with or without CKI inhibitors. Where indicated Nickel-beads were used to pull down His-tagged Ub proteins. (H–I) IB analysis of WCL derived from HeLa cells transfected with indicated constructs and treated with CHX (100 mg/L) for indicated time points (H), the relative protein levels were quantified and plotted in (I)