Skip to main content
. 2010 Sep 13;285(46):35393–35405. doi: 10.1074/jbc.M110.145052

FIGURE 1.

FIGURE 1.

LAT deletion in mature T cells impairs TCR-mediated signaling and cell proliferation. A and B, deletion of LAT in T cells. Eight to 10-week-old LATf/− and ERCre+LATf/− littermates were treated with tamoxifen. A, the efficiency of LAT deletion, as measured by GFP expression, in TCRβ+ splenocytes was examined by FACS. B, lysates of splenocytes were subjected to anti-LAT immunoprecipitation, followed by anti-LAT Western blotting. C-E, the effect of LAT deletion on T cell activation. Splenocytes were stimulated with either plate-bound anti-CD3 or P + I. Data shown are representative of at least three independent experiments. C, after overnight culture, expression of CD25 and CD69 on CD4+ T cells was examined by FACS. D, IL-2 concentrations were determined by ELISA. E, 36 h after stimulation, cells were pulsed with [3H]thymidine for an additional 6 h before scintillation counting. Triplicates were performed for each sample. F, calcium mobilization. Splenocytes were loaded with Indo-1 and then stimulated by cross-linking CD3ϵ; LATf/− CD4+ T cells (gray line); ERCre+LATf/− GFP+CD4+ T cells (black line). Data shown are representative of three independent experiments. G, biochemical analysis of TCR signaling pathways. Splenocytes were cultured with plate-bound anti-CD3ϵ plus IL-2 for 2 days and then treated with 4-OHT for 4 more days. Cells were then rested, stimulated by anti-CD3ϵ, and subsequently lysed. ZAP-70 and PLC-γ1 were immunoprecipitated and analyzed by Western blotting with anti-Tyr(P) antibody. Total protein lysates were also subjected to Western blotting with anti-Tyr(P), anti-AKT, and anti-pERK1/2 antibodies. Anti-ZAP-70, PLC-γ1, AKT, and ERK2 blots are shown as protein loading controls. Data shown are representative of four independent experiments. H, 4-OHT-treated T cells in G were rested and either left untreated (−) or stimulated with anti-CD3/CD28 (+) for 16 h. Nuclear extracts were then subjected to EMSA assay with NFκB-binding oligonucleotide. Data shown are one representative of three independent experiments.