Figure 3.

Characterizing the effects of pathogenic WAS missense mutations on WASp:Notch:NF-κB signaling module in vivo. (A) CoIP/western assay. The same gel shown in Figure 2D was sequentially reprobed with the indicated Notch signaling components. (B,D) Single and sequential ChIP-qPCR assays were performed for the indicated proteins as described in the legend to Figure 1C. (C) Left panel: Electrophoretic mobility shift assay (EMSA) with NF-κB oligo performed on the nuclei isolated from primary human CD4⁺ T_H cells, nonskewed T_H0, or T_H1- or T_H2-skewed and CD3/28-activated cells. Right panel: EMSA/Supershift assay with NF-κB oligo performed on T_H1-skewed normal T_H cells (endogenous WASp) or WAS-null T_H cells stably expressing Flag-tagged, transfected FL-WASp. The data are representative of 2 biological replicates. White arrowheads indicate location of the shifted bands.