Biochemical analysis of CCL21 costimulation. Primary mouse T cells were stimulated with anti-CD3 crosslinking, in the presence or absence of CCL21 (100 nM), for indicated times, and the activation of early signaling molecules was analyzed by immunoblotting. (A) Immunoblots of Rac2-GTP, Ras-GTP, and phosphorylated MEK1/2, ERK1/2, JNK, and Akt after costimulation of TCR and/or CCL21. For loading controls, blots were stripped and probed for total protein, or alternatively, a separate gel with lysates was analyzed. (B) Flow cytometric analysis of phosphorylated ERK1/2 formation in nonstimulated primary mouse T cells (dark gray fill) or after 5 minutes of stimulation with CCL21 (100 nM; dashed line), TCR-crosslinking (light gray fill), and TCR-crosslinking in the presence of 100 nM CCL21 (bold line). One representative experiment of 2 is shown.