(A) Representative (left) and pooled (right) results of the generation of active WASp following TCR ligation in human T cells from an HC and a DOCK8^null patient as determined by immunoblotting WASp and N-WASp CSAb immunoprecipitates with anti–WASp F-8 mAb. Aliquots from total lysates were probed for WASp to ensure equal loading and for p-ERK1/2 to verify TCR signaling. Results are representative of 2 experiments involving 2 patients and 2 controls. Quantification was performed by calculating the ratio of activated WASp in the IP to total WASp in the lysates, relative to controls. (B) Ribbon diagram of the DOCK8-CDC42 complex. Residue S1827 is in red, and the region affected by the S1827P mutation is in magenta. (C) Interaction of DOCK8 residue S1827 with CDC42 (top) and its disruption by the S1827P DOCK8^pri mutation (bottom). (D) GEF activity for CDC42 of WT DOCK8 and DOCK8^pri DHR2 domains. Relative fluorescence units (RFU) of MANT-GTP over time are shown. Results are representative of 3 experiments. (E) Representative immunoblot and quantitation of DOCK8 expression in Dock8^pri/pri and WT mice. Quantification was performed by calculating the DOCK8/GAPDH ratio relative to that in WT controls. (F and G) Representative immunoblot (F) and quantitation (G) of the association of DOCK8 with WIP and WASp in T cells from DOCK8-mutant mice. Quantification was performed by calculating the WIP/DOCK8 ratio in DOCK8 immunoprecipitates relative to that in WT controls. (H and I) Representative (H) and pooled (I) results of the generation of active WASp following TCR ligation in splenic T cells from Dock8^–/–, Dock8^pri/pri, and WT mice. The experiment was performed and the results expressed as described in A. Results in E–I are representative of 3 experiments using 3 mice from each strain. Error bars in A, E, G, and I represent the mean± SEM. *P < 0.05, by Student’s t test.