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. 2010 Oct 8;87(4):465–479. doi: 10.1016/j.ajhg.2010.08.018

Figure 5.

Figure 5

The WDR11 Interacts and Colocalizes with EMX1 In Vivo and In Vitro

(A) EMX1 was identified in the yeast two-hybrid screen as a WDR11-interacting protein. The specific interactions between these two proteins were confirmed by streaking of transformed yeast cells onto synthetic drop-out plates lacking TL (Trp/Leu) or TLH (Trp/Leu/His). Yeast AH109 cells were transformed with empty vectors (pGBKT7 and pGAD), and plasmids encoding SV40 T antigen (pTD1) and p53 (pGBKT7-p53) were utilized as a negative and positive control, respectively.

(B) Myc-WDR11 expression plasmids were transfected alone or along with HA-EMX1 into HeLa cells, the cell lysates were immunoprecipitated with anti-Myc antibody, and coprecipitated HA-EMX1 was detected via immunoblotting with anti-HA antibody.

(C) In-vitro-translated Myc-EMX1 was subjected to GST pull-down analysis with GST (lane 2) or GST-WDR11 (lane 3). The bound proteins were detected via immunoblotting with anti-Myc antibody.

(D) HA-EMX1 and GFP-WDR11 expression plasmids were transfected into U2OS cells, and then cells were treated with leptomycin B (LMB), an inhibitor of nuclear export. Fluorescence microscopy analysis helped determine localizations of HA-EMX1 and GFP-WDR11. Nuclei were stained with DAPI.

(E) Wild-type Myc-WDR11 and its deletion mutants were synthesized in vitro and subjected to a GST pull-down assay with GST (center panel) or GST-WDR11 (right panel). The N, M, and C denote the WDR11 N terminus (amino acids 1–361), middle portion (amino acids 362–830), and C terminus (amino acids 831–1224), respectively. The positions of missense mutations found within the N terminus and central region of WDR11 in IHH patients are marked as m1 through m4 on the schematic diagrams of WDR11.

(F) The wild-type and missense mutant WDR11 expression plasmids were transfected into HEK293 cells along with HA-EMX1 expression plasmids. The cell lysates were immunoprecipitated with anti-Myc antibody, and the association of EMX1 with wild-type WDR11 or missense mutants was determined via immunoblot analysis with anti-HA antibody.