Defective TCR signaling and calcium influx in RhoH-null thymocytes. Thymocytes of 2-month-old mice were stimulated with biotinylated antibodies CD3 (C) or CD3 and CD4 (A-B, D) and streptavidin for 5 minutes at 37°C. Phosphorylation of ZAP70(Y319)/Syk(Y352) (A), lck(Y505) (B), Erk1/2(T202/Y204) (C), and p38 MAPK (T180/Y182) (D) was measured by FACS. Different thymocyte populations were distinguished (filled, nonstimulated; line, stimulated). Differences between the mean of stimulated and nonstimulated (specific mean) cells are shown. *P < .05; **P < .01. n [control]/RhoH^−/−]: 5/5 (A-B, D). n [control]/RhoH^−/−]: 4/5 (C). Thymocytes (E) or splenocytes (F) of 2-month-old mice were loaded with Fluo-4 and stained on ice for CD4, CD8, and CD3. After warming to 37°C, baseline Fluo-4 fluorescence was determined, and TCR signaling was induced by cross-linking CD3 with streptavidin. Presented is the percentage of cells above a threshold fluorescence (responding cells; left panel) and the mean fluorescence of all cells (right). Shown are representative results of 5 (E) or 2 (F) independent experiments. Error bars indicate the standard deviation.