Supporting Figures

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Fig. 5. (A) Semiquantitative RT-PCR detection of Cav b4 subunit expression in CD4 T cells. Purified CD4 T cells were left unstimulated (day 0) or were stimulated with plate-bound anti-CD3 plus anti-CD28 Abs for the indicated period. Brain cDNA was used as positive control. (B) Genotyping b4 mutant mice. PCR of tail-extracted DNA was performed to detect the 4-nt insertion in the b4 mutant (mut) mice relative to WT.

Fig. 6. Normal T cell development of the b4 mutant and b3 knockout mice. (A) Thymic development analysis in 10- to 14-day-old b4 mutant mice and WT littermate. (B) Thymic development analysis in WT and b4 mutant bone marrow chimera mice. WT and b4 mutant bone marrow cells were injected i.v. into irradiated RAG1^-/- mice. Four weeks later, thymus was analyzed by using appropriate Abs and flow cytometry. (C) Similar expression of activation markers by b4 mutant mice and WT littermate in the periphery. Spleen and lymph node cells were stained for different activation markers. Surface expression of CD4, CD8, CD69, CD25, CD62L, and CD44 was subsequently assessed by flow cytometry. (D) Thymic development analysis in WT and b3 knockout mice.

Fig. 7. (A) Comparison of peak and plateau calcium responses of b4 mutant T cells and control littermate after TCR stimulation. (B) Comparison of fluo-3/AM loading in T cells from b4 mutant mice and WT littermate assessed using flow cytometry. (C) TCR and thapsigargin-mediated calcium response by b4 mutant T cells and WT littermate in calcium-free medium (0 mM Ca²⁺). Ionomycin was added at the end of each experiment as a positive control in calcium-containing medium (1 mM).

Fig. 8. (A) Proliferation of b4 mutant CD4 T cells stimulated by plate-bound anti-CD3 plus anti-CD28 Abs assessed by CFSE. (B) CD4 T cells were stimulated as in A in the presence of exogenous human IL-2 (20 units/ml). IL-2 production was evaluated 24 h later by ELISA.

Fig. 9. CD4 T cells from WT or b4 mutant bone marrow chimeras were stimulated for 4 days, washed, and then restimulated for an additional 24 h. Cytokine production was measured by ELISA.

Fig. 10. The depolarization of T lymphocytes is nifedipine-sensitive. Depolarization was assessed with the voltage-sensitive dye DiBAC₄(3) by using flow cytometric analysis after stimulation (via TCR or with KCl) of cells that have been pretreated or not with 2 mM nifedipine.