(A) Western blotting analysis of B-Raf expression in thymocytes fromRAG2−/− mice, DP and SP thymocytes sorted from C57BL/6 mice, peripheral (splenic) T cells fromRAG2−/− chimeras reconstituted with B-Raf+/+, B-Raf +/− or B-Raf−/− FL cells, as well as mock- and B-Raf-transfected Jurkat cells. Raf-1 and ERK blottings are shown as loading controls. PC12 and NIH3T3 were utilized as positive and negative controls of B-Raf expression, respectively. (B, C) In vitro kinase assays for Raf-1 and B-Raf isolated from thymocytes stimulated with 10 µg/mL anti-CD3 Ab or 25 ng/mL PMA for 10 min (B) or anti-CD3Ab for the indicated times (C). Recombinant GST-MEK was used as a substrate, and incorporated 32P radioactivity was visualized by autoradiography (upper left panel). ERK phosphorylation was determined by blotting with an antibody specific for phosphorylated ERK. Equal loading of each Raf isoform and ERK was confirmed (lower left panels). (D) Splenic T cells were stimulated with 5 µg/mL anti-CD3 Ab for the indicated times in an in vitro kinase assay for B-Raf and Raf-1 performed as described in (B).