Figure S3.
Additional supporting data related toFigs. 3and4. (A) Gene targeting strategy and genotyping of tail DNA from mice with indicated genotypes. (B) Immunoblot analysis of VMP1 expression in T cells from mice with indicated genotypes. (C) Thymi from mice with indicated genotypes were analyzed by flow cytometry. (D) TMRM level in T cells from mice with indicated genotypes was examined by flow cytometry. (E and F) Mice with indicated genotypes were infected with L. monocytogenes expressing OVA (LM-OVA). CD8 T cell response was analyzed on day 8 after infection. (E) Representative plots and statistics of OVA257-264-H2Kb tetramer-positive cells in spleen are shown. Plots were gated on single live cells. (F) Representative plots and statistics of IFNγ-positive cells after ex vivo stimulation with OVA257-264 peptide (SIINFEKL) are shown. Plots were gated on CD8α+ cells. (G) pLJM1- or pCMV-based expression system. (H) HEK293T cells were transfected with indicated plasmids, and the expression of VMP1 was examined by immunoblot. (I) ER Ca2+ in HEK293T cells transfected with indicated plasmids was measured by flow cytometry. (J) The expression of indicated proteins in HEK293T cells or VMP1-KO HEK293T cells stably expressing empty vector, VMP1, or VMP1-D272N was analyzed by immunoblot. (K) gDNA sequence of Vmp1 loci from WT and Vmp1D272N/+ mice. Nucleotides encoding 272D (GAC) and 272N (AAT) are labeled with green and red colors, respectively. Arrows indicate double peaks of G/A and C/T reads from sequencing result. (L) PCR genotyping of WT and Vmp1D272N/+ mice. (M) Immunoblot analysis of VMP1 expression in T cells from mice with indicated genotypes. (N) ER Ca2+ stored in T cells from mice with indicated genotypes was examined by flow cytometry. (B, H, J, and M) Representative blots from one of three independent experiments. (C–F, I, and N) Representative plots and statistics from one of three independent experiments. n = 4 mice per genotype in C and D, n = 3 mice per genotype in E, F, I, and N; each symbol represents an individual mouse; data represent mean ± SEM, two-tailed unpaired t test in C–F and N, one-way ANOVA in I; * <0.05, ** <0.01, *** <0.001, **** <0.001. Source data are available for this figure: SourceData FS3.

Additional supporting data related to Figs. 3 and 4 . (A) Gene targeting strategy and genotyping of tail DNA from mice with indicated genotypes. (B) Immunoblot analysis of VMP1 expression in T cells from mice with indicated genotypes. (C) Thymi from mice with indicated genotypes were analyzed by flow cytometry. (D) TMRM level in T cells from mice with indicated genotypes was examined by flow cytometry. (E and F) Mice with indicated genotypes were infected with L. monocytogenes expressing OVA (LM-OVA). CD8 T cell response was analyzed on day 8 after infection. (E) Representative plots and statistics of OVA257-264-H2Kb tetramer-positive cells in spleen are shown. Plots were gated on single live cells. (F) Representative plots and statistics of IFNγ-positive cells after ex vivo stimulation with OVA257-264 peptide (SIINFEKL) are shown. Plots were gated on CD8α+ cells. (G) pLJM1- or pCMV-based expression system. (H) HEK293T cells were transfected with indicated plasmids, and the expression of VMP1 was examined by immunoblot. (I) ER Ca2+ in HEK293T cells transfected with indicated plasmids was measured by flow cytometry. (J) The expression of indicated proteins in HEK293T cells or VMP1-KO HEK293T cells stably expressing empty vector, VMP1, or VMP1-D272N was analyzed by immunoblot. (K) gDNA sequence of Vmp1 loci from WT and Vmp1D272N/+ mice. Nucleotides encoding 272D (GAC) and 272N (AAT) are labeled with green and red colors, respectively. Arrows indicate double peaks of G/A and C/T reads from sequencing result. (L) PCR genotyping of WT and Vmp1D272N/+ mice. (M) Immunoblot analysis of VMP1 expression in T cells from mice with indicated genotypes. (N) ER Ca2+ stored in T cells from mice with indicated genotypes was examined by flow cytometry. (B, H, J, and M) Representative blots from one of three independent experiments. (C–F, I, and N) Representative plots and statistics from one of three independent experiments. n = 4 mice per genotype in C and D, n = 3 mice per genotype in E, F, I, and N; each symbol represents an individual mouse; data represent mean ± SEM, two-tailed unpaired t test in C–F and N, one-way ANOVA in I; * <0.05, ** <0.01, *** <0.001, **** <0.001. Source data are available for this figure: SourceData FS3.

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