Pep−/− and CD45 E613R alleles influence TCR signaling in a developmental stage-specific manner. (A) Graph depicts mean fluorescence intensity (MFI) for CD5 staining on gated double positive (DP) thymocytes. Values are the mean of three biological replicates +/− SEM and represent at least 5 independent experiments each involving 3 animals/genotype. (B) Intracellular calcium levels of Fluo-4 AM-loaded thymocytes (gated for small size to identify DP population). Green fluorescence was monitored by flow cytometry before and after stimulation with soluble anti-CD3ε antibody in the upper panel or ionomycin in the lower panel. Genotypes are identified as in Figure (A). Plots are representative of at least 4 independent experiments (C) DP thymocytes were stimulated with soluble anti-CD3ε. Fixed and methanol permeabilized cells were then stained with antibody against phospho-Erk and assessed by flow cytometry. Histograms represent gated DP thymocytes. Wild type DP thymocytes are represented by the shaded gray histogram in each plot for comparison. PMA responses in all genotypes were identical. Plots are representative of at least 5 independent experiments. (D) LN T cells were stimulated with anti-CD3 antibody, fixed and permeabilized with methanol and subsequently stained with antibodies to CD4, CD8, CD44, and phospho-Erk. Histograms represent staining for phospho-Erk on cells gated for CD4, CD8 and either CD44 low or high surface staining to identify naïve and memory subsets. Shaded histograms represent wild type for comparison. Plots are representative of at least three independent experiments each with three biological replicates /genotype. (E) In vitro generated effector CD4 or CD8 T cells were stimulated, fixed, and permeabilized as described above, and subsequently stained with antibody to phospho-Erk. Histograms represent staining for phospho-Erk. Shaded histograms represent wild type for comparison. Plots are representative of two independent experiments.