1. WDR11 forms a tertiary complex with EMX1 and GLI3
WDR11 forms a tertiary complex with EMX1 and GLI3 Co‐immunoprecipitation (co‐IP) analyses of HEK293 cells co‐transfected with 6xMyc‐tagged WDR11 and HA‐tagged EMX1 expression constructs either alone or together with GLI3 full‐length (GLI3FL) expression construct, demonstrating the formation of WDR11‐EMX1‐GLI3FL tertiary complex (left panel). In a reciprocal co‐IP experiment, 6xMyc‐tagged GLI3R was co‐transfected with HA‐tagged EMX1 either alone or together with untagged WDR11 expression construct to demonstrate the formation of GLI3R‐EMX1‐WDR11 complex. The disease‐associated mutant WDR11 showed a reduced binding capacity to EMX1 within this complex (right panel).Co‐IP analyses of mouse testes tissue lysates showed that endogenous WDR11 could bind to endogenous GLI3FL (190 kDa), GLI3R (83 kDa) and EMX1/2 (35 kDa) proteins. Tissue lysates were precipitated with anti‐WDR11 antibody or non‐immune IgG, and probed with antibodies against GLI3, WDR11 or EMX1/2.6xMyc‐GLI3R and HA‐EMX1 were co‐transfected in WT or Wdr11−/− MEFs and precipitated with anti‐Myc antibody and probed with EMX1 and GLI3 antibodies. EMX1‐GLI3 complex did not form in Wdr11−/− MEFs, indicating that EMX1‐GLI3 failed to bind in the absence of WDR11.The relative expression ratios of endogenous GLI3FL/R normalized to the loading control (β‐actin) were assessed by Western blot in HEK293 cells after WDR11 overexpression (left panel), in MEFs of WT and Wdr11−/− embryos (middle panel) and in tissue lysates of WT and Wdr11−/− kidney (right panel). Band intensities of GLI3FL and GLI3R were quantified by ImageJ. Source data are available online for this figure.
Yeon‐Joo Kim et al. EMBO Rep. 2018;19:
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