Figure 5. Reduced Calcium flux in DP thymocytes from 1017-DRAK2 Tg mice stimulated with anti-CD3 and anti-CD4, but not in SP thymocytes.

A To detect alteration in Calcium signaling upon TCR stimulation by flow cytometry, thymocytes labeled with Fluo-3 and Fura red and stained with CD4 and CD8 were incubated with biotinylated antibodies to CD3 and CD4 before cross-linking with streptavidin as described. Black lines indicate Calcium flux in DRAK2 transgenic thymocytes, grey lines represent Wt controls. For FACS analysis, thymocytes were first sampled for ~60 s to establish baseline Ca²⁺-levels before stimulation with streptavidin (arrow “SA”). After ~480 s of stimulation, maximal Calcium release was induced by addition of ionomycin (arrow “iono”). Data represent the kinetics of calcium mobilization in response to stimulation in total thymocytes, DP thymocytes and CD⁴⁺ thymocytes plotted as ratio of FL-1 (Fluo-3) to FL-3 (Fura red). B Unaltered signalling in 1017-DRAK2 Tg thymocytes in response to TCR crosslinking. Total thymocytes were incubated with the indicated amounts of biotinylated anti-CD3 plus biotinylated anti-CD4, followed by crosslinking for 2 min. with streptavidin. Western blotting was performed on lysates using anti-phospho- and total-Erk1/2, anti-phospho- and total-Jnk1/2, anti-phospho-Tyrosine, anti-DRAK2 and anti-α-tubulin as a loading control. C Detection of calcium signalling as performed in A. on total splenocytes, except that incubation was with anti-CD3 alone before crosslinking with streptavidin as described. D Altered signalling in 1017-DRAK2 Tg splenocytes in response to TCR crosslinking. Total splenocytes were incubated with the indicated amounts of biotinylated anti-CD3 +/- biotinylated anti-CD28, followed by crosslinking for 2 min. with streptavidin. Western blotting was performed as in B.