Kumar et al. 10.1073/pnas.0509176102. |
Fig. 8. Expression of PLC-g1, Itk, and Lck in Jurkat cells and J14 cells transfected with WT SLP-76 or SLP-76D185–194. Shown is Western blot analysis of cell lysates; b-actin was used to assess equivalent loading.
Fig. 9. Impaired NFAT activation in SLP-76 mutant T cells. Jurkat T cells were stimulated with plate-bound anti-CD3 antibodies for the indicated time periods in the presence of 2 mM CaCl2. Total cellular extracts were subjected to SDS/PAGE, and immunoblots were incubated with anti-NFAT1 antibodies. Bands representing phosphorylated (P) and dephosphorylated (deP) NFAT1 are indicated by arrows. b-actin was used to assess equivalent loading.
Fig. 10. FACS analysis of thymocytes and splenocytes derived from a second founder SLP-76–/– mice reconstituted with SLP-76-D185–194 transgene. (A) Surface expression of CD4 vs. CD8 on total thymocytes. (B) TCRb expression on total thymocytes. (C and D) Surface expression of CD4 and CD8 (C) and CD3 (D) on spleen cells.
Fig. 11. Apoptosis in thymocytes of SLP-76-D185–194 mice is not rescued by bcl-2. (A) Annexin V staining of double and single positive thymocytes in SLP-76 WT, SLP-76D185–194, and SLP-76D185–194/bcl-2 mice. (B) FACS analysis of intracellular expression of bcl-2 transgene in thymocytes of SLP-76D185–194/bcl-2 mice. (C) CD4 and CD8 staining in thymocytes of SLP-76D185–194/bcl-2 mice. The results are representative of at least three experiments.
Supporting Text
Purified T cells from SLP-76+/+ and SLP-76–/–D185–194 mice were loaded with 1 mM fura-2 acetoxymethyl ester (Molecular Probes) for 30 min at room temperature, washed, and resuspended in loading medium (RPMI medium 1640/10% FCS). Cells were incubated with 5 mg/ml biotinylated anti-CD3 (145-2C11, Pharmingen) for 15 min at room temperature and attached to poly(L-lysine)-coated coverslips mounted in a RC-20 closed bath chamber (Warner Instruments). Cells were perfused in 2 mM calcium Ringer's solution (155 mM NaCl/4.5 mM KCl/10 mM D-glucose/5 mM Hepes, pH 7.4/1 mM MgCl2/2 mM CaCl2). For stimulation, anti-CD3 was crosslinked by using 10 mg/ml streptavidin and 0.5 mM ionomycin (Calbiochem), respectively. Single-cell video imaging was performed on an S100 inverted epifluorescence microscope (Zeiss) by using OPENLAB imaging software (Improvision). Fura-2 emission was detected at 510 nm after excitation at 340 and 380 nm, respectively, with ratios of 340/380 being calculated for each 5-sec interval after background subtraction. Calibration values (Rmin, Rmax, and Sf) were derived from cuvette measurements with a calcium calibration buffer kit (Molecular Probes) as described in ref. 1. For each experiment, ≈200 cells were analyzed.
1. Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440–3450.