FIG. 2.
Knockdown of RasGRP1 affects induced Ras-ERK activation more profoundly than knockdown of SOS1. (A) siRNA duplexes for mouse (mo. [control]) or human (Hu.) SOS1 were introduced via transient transfection into wild-type Jurkat or mutant JPRM441 cells. The resultant populations were stimulated 40 h after transfection by cross-linking the stably expressed CD25ζζ and analyzed as in Fig. 1. The relative percentages of SOS1 and RasGRP1 expression as well as ERK phosphorylation were determined by normalizing for Ras expression. Both Jurkat and JPRM441 cells express similar levels of RasGRP3 and SOS2 (data not shown). α-CD25, anti-CD25. (B) Wild-type Jurkat cells were transfected with the indicated siRNA duplexes. Forty hours later, cells were stimulated by cross-linking the TCR and analyzed by Western blot analysis as in panel A, normalizing for Grb2 expression. α-TCR, anti-TCR. (C) Mouse Rasgrp1 (control), RasGRP1, and SOS1 siRNA duplexes were introduced into purified human CD4+ T cells using an Amaxa electroporator. Forty hours later, cells were stimulated by cross-linking the TCR and CD4. Expression of the indicated proteins, as well as phosphorylation of ERK kinases, was determined by Western blot analysis (the same samples were run on two separate gels, analyzing SOS1 and Grb2 on one gel and RasGRP1, P-ERK, and Grb2 on the other). The percentage of ERK activation and RasGRP1 and SOS1 expression levels were determined by normalizing for Grb2 expression. α-CD4, anti-CD4. (D) Expression levels of SOS1 and RasGRP1 were compared between human CD4 T cells and the Jurkat T-cell clone by combining the primary antibodies into one mixture. Grb2 normalizes for the amount of protein loaded.