VMP1 deficiency results in ER Ca ²⁺ overload and naive T cell apoptosis. (A) ER Ca²⁺ stored in T cells from mice with indicated genotypes was examined by flow cytometry. Representative plots and statistics are shown. (B) Ca²⁺ influx triggered by CD3ε crosslinking in T cells from mice with indicated genotypes was examined by flow cytometry. T cells were incubated with 10 μg/ml anti-CD3-biotin, then 10 μg/ml streptavidin (STV) was used for cross-linking. (C) Absolute number of T cells from spleen (SPL) and peripheral lymph nodes (pLN) of mice with indicated genotypes. Mice were 4–6 wk old. (D) Apoptosis of freshly isolated T cells from mice with indicated genotypes was analyzed by flow cytometry. (E) Caspase-3 staining of freshly isolated T cells from mice with indicated genotypes was analyzed by flow cytometry. (F) Experimental setup for BM chimera experiments. (G) Flow cytometry analysis of donor-derived cells before transfer and 8 wk after transfer. (H) Cells from SPL and pLN of mice with indicated genotypes were analyzed by flow cytometry. (A, C–E, G, and H) Representative plots and statistics from one of two independent experiments. (B) Representative plots from one of three independent experiments. n = 5 mice per genotype in A, n = 3–4 mice per genotype in C, n = 4 mice per genotype in D and E, n = 6 mice per genotype in G, n = 3 mice per genotype in H; each symbol represents an individual mouse; data represent mean ± SEM, two-tailed unpaired t test; * <0.05, ** <0.01, *** <0.001, **** <0.001.