Impaired TCR signaling in RhoH-null DP thymocytes. DP thymocytes of 4- to 8-week-old mutant mice were sorted by FACS or MACS microbeads. TCR signaling was induced as indicated by cross-linking of biotinylated CD3 and CD4 antibodies with streptavidin for 5 minutes (A-E, I), 30 seconds (G), or indicated times (F, H) at 37°C. Total lysates were analyzed by Western blot for ZAP70-P319 (A), LAT-P195 (C), PLCγ1-P783 (D), Vav2-P (F), and Erk-P (I). Immunoprecipitations (IPs) of LAT (B) and Vav1 (E) were blotted with antiphosphotyrosine antibodies and reprobed with LAT (B) or Vav1 (E). Amounts of active Rac1 and Rac2 were determined by pull-down assays (G-H). Bar graphs represent quantifications of 5 (A), 3 (B-D,I), 4 (E), 5 (F), 10 (G), or 4 (H) independent experiments. *P < .05; **P < .01. All values are normalized to equal total amounts of the corresponding protein determined by Western blot. Representative examples of the Western blots are shown in Figure S9. Error bars show the standard deviation.