FIGURE 2.

Enhanced ERK phosphorylation in DGKζ-deficient CD8⁺ T cells. A, 1 × 10⁶ MACS-purified wild type or DGKζ^−/− CD8⁺ T cells were incubated with α-CD3 (500A2, 2.5 μg/ml) for the indicated duration, or with DAG analog (phorbol myristate acetate (P), 1 μg/ml) for 10 min. Cell lysates from these reactions were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the indicated primary antibody. A representative blot is depicted from one of three experiments. PLCγ1, phospholipase Cγ1. B, 2 × 10⁶ wild type or DGKζ^−/− splenocytes were incubated with or without 2.5 μg/ml α-CD3 (500A2, flow cytometry plots) or varied concentration of α-CD3 (right panels), for 15 min prior to fixation and permeabilization. Cells were stained with fluorochrome-conjugated antibodies for cell surface proteins CD4, CD8, and CD44 and intracellular pERK and subjected to flow cytometry. Left panels, flow cytometry plots of pERK-Ax488 (y axis) by CD44-PE (x axis) in CD8⁺ gated cells. Right panels, analysis of flow cytometry data over indicated concentrations of α-CD3 for: % pERK positive cells (top panel), mean fluorescent intensity of pERK per positive cell (middle panel), or % pERK-positive cells normalized to the 100% maximum observed per genotype (bottom panel). C, 2 × 10⁶ CD45.1⁺ splenocytes were loaded with 10 μm OVAp (SIINFEKL), altered peptide variants A2, Y3, Q4, T4, V4, or no peptide (none) prior to incubation with 1 × 10⁶ CD45.1- OT-I, or OT-I splenocytes for 15 min. Cells were processed as in C and CD45.1⁻ CD4⁻ CD8⁺ cells were evaluated for the percentage of pERK-positive cells (left panel) or mean fluorescent intensity of pERK per cell (right panel).