(A and B) Representative immunoblot and quantitative analysis of co-IP of WIP and DOCK8 from peripheral blood T cell lysates from an HC and a WASp^null patient (A) and of mouse splenic T cells from WT and Was^–/– mice (B). T cells were examined 0 and 10 minutes after (+) anti-CD3 stimulation. Using densitometric scanning, quantification of the results was performed by calculating the ratio of WIP/DOCK8 bands in the DOCK8 immunoprecipitates to that in the lysates relative to controls. The results in A represent 2 WAS patients and 2 controls examined in 2 independent experiments, and in B, 3 WT and 3 Was^–/– mice were examined in 3 independent experiments. Aliquots from the lysates used for IP were probed with MALT1 in A and with GAPDH in B to ensure equal loading. Error bars in A and B represent the mean ± SEM. Student’s t test. (C) Co-IP of rWIP-EGFP, but not EGFP, with rMyc-tagged DOCK8 (DOCK8-Myc) proteins (top panel) and of rDOCK8-Myc with rWASp-Flag, but not rMALT1-Flag, in the presence, but not in the absence, of rWIP-EGFP (bottom panel). (D) Map of the WIPΔWBD protein. (E) Representative immunoblot of the co-IP of WIP-EGFP and WIPΔWBD-EGFP with Myc-tagged DOCK8 in 293T cell transfectants. LRRC8A-Myc and EGFP transfectants were used as negative controls, and lysate aliquots were probed for Myc and EGFP to ensure equal loading. The * denotes a non-specific band. (F) Co-IP of DOCK8 and WASp with WIP in human T cells, using the 3D10 and D12C5 anti-WIP mAbs directed against distinct epitopes in the N-terminus and C-terminus of WIP, respectively. Data in C, E, and F represent 4 independent experiments.