RasGRP1 in T cells is subject to PKC-dependent regulatory phosphorylation on Thr184, while RasGRP4 is regulated by other means. (A) Jurkat T cells were left untreated or were stimulated with anti-TCR antibodies (OKT3) for 5 minutes or PMA for 10 minutes, and lysates were probed for Thr184-phosphorylated RasGRP1 and total RasGRP1 (top). Signaling to Erk was also monitored (bottom). (B) Jurkat T cells were pretreated with PKC inhibitors and stimulated with the anti-TCR antibody OKT3. Lysates were assayed for phospho-RasGRP1 and total RasGRP1, as in panel A. Lysates were also probed for phospho-Erk to monitor signaling. (C) Mouse T cells were pretreated with inhibitors and stimulated with anti-TCR antibodies (2C11) or PMA, and lysates were probed for signaling molecules as in panel B. Numbers are quantification of band intensity. (D) Rat2 cells expressing empty vector pBabeHygro (Hygro) or RasGRP4 in the same vector were pretreated with inhibitors, followed by PMA treatment, as indicated. Total cell lysates were blotted for phospho-Erk to monitor Ras-Erk signaling and total Erk (tErk) as a loading control. Numbers are quantification of band intensity. Results are representative of at least 2 independent experiments.