Figure 1.  The GBD of WASp is not essential for WASp activity. (A) Scheme that shows the domain organization of wild-type and a GBD-deleted (WASpΔGBD) WASp cDNA used to derive WAS−/−ΔGBD mice. Positions of all tyrosine residues within WASp are indicated. (B) Lysates obtained from WASp or WASpΔGBD-expressing Cos-7 cells were incubated with GTPγS-loaded GST-cdc42 fusion protein bound to glutathione sepharose beads and the complexes were then resolved by SDS-PAGE and sequential immunoblotting analysis with anti-WASp and anti-cdc42 antibodies (top two panels). Immunoblotting analysis demonstrating WASp and WASpΔGBD expression in Cos-7 transfectants (Tfn) is shown in the bottom panel. (C) Immunoblotting analysis showing the WASp species detected in thymocytes and lymphocytes from wild-type (WT), WAS−/−, and WAS−/−ΔGBD mice using an anti-WASp antibody. (D) Lymphocytes and thymocytes isolated from 4–8-wk-old wild-type (WT), WAS−/−, and WAS−/−ΔGBD mice were cultured for 48 h in medium alone or with either 1 μg/ml anti-CD3 antibody or 0.2 μg/ml anti-CD3 plus anti-CD28 antibody. Antigen receptor–evoked proliferative responses were determined after an 18-h pulse with [3H]thymidine. Values represent the means (± SEM) of four independent experiments. (E) Thymocytes from wild-type (WT), WAS−/−, and WAS−/−ΔGBD mice were isolated and stimulated with anti-CD3 plus anti-CD28 antibodies for 30 min on ice followed by cross-linking with a secondary antibody. Cells were fixed with 5% paraformaldehyde and F-actin content was quantified by flow cytometric analysis of FITC phalloidin–stained resting (bold lines) and stimulated (dashed lines) cells. The results are representative of three independent experiments.