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

Figure 1.

Figure 1

ZNRF3 protein level is regulated by Cullin 1 based E3 ligases, in a 26S proteasome dependent manner. (A and B) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HeLa cells transfected with WT or RING-domain deleted (ΔRING) ZNRF3. Resulting cells were treated with MG132 (10 μmol/L) (A) or MLN4924 (10 μmol/L) (B) for 12 h before harvesting for IB analysis. (C–E) IB analysis of immunoprecipitations (IP) and WCL derived from HeLa cells co-transfected ZNRF3 with various Cullin family proteins (C), Skp1 (D) or Rbx1 (E) after treated with MG132 (10 μmol/L) for 12 h before being harvested for IB analysis. (F) IB analysis of WCL derived from U2OS cells lentivirally infected with control (sh-Scr) or multiple independent shRNAs against Cullin 1 (sh-Cullin1). Infected cells were selected with 1 μg/mL puromycin for 72 h to eliminate non-infected cells before harvesting. (G and H) IB analysis of WCL derived from the cell lines in (F) treated with CHX (100 mg/L) for different time point (G), relative protein levels were quantified and plotted in (H)