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. 2018 Jan 29;37(4):e97311. doi: 10.15252/embj.201797311

Figure 2. Evi stabilization is dependent on Wnt palmitoylation.

Figure 2

  1. Schematic illustration of the Porcn‐mediated Wnt palmitoylation, which is important for Evi‐Wnt interaction and which is blocked upon Porcn inhibition (LGK974), in PorcnKO cells and by using a palmitoylation‐deficient S209A Wnt3A mutant.
  2. Wild‐type or stable Wnt3‐and Wnt5B‐expressing HEK293T cells were treated with 5 μM LGK974 for 48 h and subjected to Western blot analysis.
  3. Western blot analysis of endogenous Evi in wt, PorcnKO, or EviKO HEK293T cells upon overexpression of Wnt3A or IGFBP5‐V5. PorcnKO1.2 and PorcnKO1.4 indicate clone #2 and clone #4 of PorcnKO HEK293T cells generated with Porcn sgRNA1 (Appendix Fig S3). Clonal EviKO HEK293T cells were generated with Evi sgRNA2 (EviKO2.9; clone #9) or Evi sgRNA1 (EviKO1.1; clone #1; Appendix Fig S2). Increase in total β‐catenin protein served as control for Wnt pathway activation.
  4. Western blot analysis of endogenous Evi in HEK293T cells transfected with the indicated overexpression plasmids. When indicated, the cells were additionally treated with 5 μM LGK974 for 48 h.
  5. Western blot analysis of endogenous Evi in HCT116 or A375 cells treated with 5 μM LGK974 or DMSO for the indicated hours (h). All Western blots are representative of three independent experiments. β‐Actin was used as a loading control, LRP6 as a reference membrane protein and EGF‐Myc and IGFBP5‐V5 as controls for secreted proteins. Specific Evi bands are indicated by arrows, and unspecific bands are marked by asterisks.
  6. Scheme: Wnt‐induced Evi stabilization is blocked in the absence of Wnt palmitoylation (PorcnKO, LGK974, Wnt3A S209A).

Source data are available online for this figure.