Ionomycin-up-regulated GRAIL expression is associated with reduced CD40L expression. A and B, MACS-purified CD4 T cells from BALB/c mice were anergized with a low (0.1 μM), medium (0.5 μM), or high (1.5 μM) ionomycin dose with or without PMA (200 ng/ml) overnight. Cells were then harvested for QPCR (A) or Western blot analysis (B) of GRAIL expression. N.S., nonspecific band used as a loading control; IB, immunoblot; Norm., normalized. C, MACS-purified BALB/c CD4 T cells were anergized with 0 μM (left) or 1.5 μM (right) ionomycin overnight. Live cells were then washed, stimulated, and stained for cell surface CD40L. D, Histograms representing the percentage of cell surface CD40L expressed on gated CD4 T cells with varying conditions of ionomycin anergy treatment and recall stimulation. Black histogram represents CD4 T cells treated with 0 μM ionomycin, dark gray histogram denotes CD4 T cells treated with 0.5 μM ionomycin, and light gray histogram represents CD4 T cells treated with 1.5 μM ionomycin.