Effect of missense variants in the BEACH domain identified in GPS cases on the interaction with Dock7 and Vac14. (A) Diagram representing the construct PB-FTAP including the PH and BEACH domains of Nbeal2 and the FTAP-tag. (B) HEKs whole cell lysates containing a mixture of PB-FTAP and PBW-FTAP in a 1:1 ratio, or only PB-FTAP and PBW-FTAP (input) were immunoprecipitated using an isotype control IgG (Ct-IgG) or anti-FLAG. (C) Diagram representing the functional domains of Nbeal2 as per Figure 1A. Six amino acid mutations causative of GPS were selected on basis of their predicted functional consequences. (D) Computer model of the BEACH domain illustrating the 6 mutations shown in panel C and color-classified by disruptive effect. (E) PBW-FTAP in HEKs expressing the reference allele or the 6 mutations were lysed (input), IP with anti-FLAG and blotted with anti-Vac14. Quantitation of the interaction Vac14-Nbeal2 for each of the variants and control was achieved by densitometry. (F) Similar assays performed in panel E, but immunoblots (IBs) were developed with anti-Dock7. Experiments were performed on 5 independent occasions. Bars represent mean ± SEM. *P value < .05.