Supplementary Figure 3: G131D or E does not impact activation of ZAP-70 and Lck.
From: Slow phosphorylation of a tyrosine residue in LAT optimizes T cell ligand discrimination
a. Immunoblot analysis of J.LAT.WT, J.LAT.G131D, or J.LAT.G131E cells stimulated with a range of titrated anti-CD3 for one minute at 37oC. Lysates were prepared and run on 12% NuPage Bis-Tris protein gels, and subjected to immunoblot analysis with various anti-pY or anti-total protein as indicated. Data are representative of at least six independent experiments. b. Bar graphs depicting the fold change of phospho-tyrosines of specific proteins (as indicated) of J.LAT.WT, J.LAT.G131D, or J.LAT.G131E cells following stimulation with titrated concentrations of anti-CD3. Each symbol represents the analysis of one experiment. The lines above the bar graphs represent the significance of standard deviations (mean; n = 6 for Lck p-Y394, ZAP-70 p-Y319, LAT p-Y191, LAT p-Y226; n =7 for ZAP-70 Y493; n = 8 for LAT p-Y171; n = 4 for ζ p-Y142). ns = not significant. For Lck p-Y394, WT vs D from left to right: P = 0.2751; P = 0.9989; P = 0.9923; P = 0.9933; P > 0.9999; WT vs E from left to right: P > 0.9999; P = 0.9847; P = 0.9565; P = 0.9933; P > 0.9999; P = 0.9987; For ζ p-Y412, WT vs D from left to right: P > 0.9999; P = 0.9378; P = 0.9988; P = 0.9996; P = 0.9932; WT vs E from left to right: P > 0.9999; P = 0.9119; P > 0.9999; P > 0.9999; P > 0.9999; For ZAP-70 p-Y319, WT vs D from left to right: P > 0.9999; P = 0.9958; P = 0.7575; P = 0.6990; P = 0.1022; WT vs E from left to right: P = 0.5233; P = 0.5144; P = 0.4558; P = 0.3149; P = 0.9968; For ZAP-70 p-Y493, WT vs D from left to right: P > 0.9999; P = 0.9180; P = 0.4685; P = 0.3701; P = 0.9789; WT vs E from left to right: P = 0.8176; P > 0.9999; P = 0.9429; P = 0.7203; P > 0.9999; For LAT p-Y171, WT vs D from left to right: P = 0.2263; P > 0.9999; P = 0.9997; P = 0.9986; P = 0.7571; WT vs E from left to right: P = 0.9852; P = 0.9801; P > 0.9999; P = 0.9994; P = 0.9983; For LAT p-Y191, WT vs D, from left to right: P = 0.9995; P = 0.6763; P = 0.9994; P = 0.9999; P > 0.9999; WT vs E, from left to right: P = 0.9998; P = 0.9996; P > 0.9999; P = 0.9998; P = 0.9998; For LAT p-Y226, WT vs D, from left to right: P > 0.9999; P = 0.9984; P > 0.9999; P > 0.9999; P = 0.9869; WT vs E, from left to right: P = 0.3200; P = 0.9887; P = 0.9985; P > 0.9999; P > 0.9999. One-way ANOVA test. c. Bar graphs depicting the fold change of LAT p-Y132 or PLC-γ1 p-Y783 of J.LAT.WT, J.LAT.G131D, or J.LAT.G131E cells following stimulation with titrated concentrations of anti-CD3. The relevant bands in each immunoblot were quantified by Image Lab. The signal intensity of LAT p-Y132 or PLC-γ1 p-Y783 was normalized to the total protein (LAT or PLC-γ1) first and then normalized to the 0 sec time point of J.LAT.WT cells’ response. The experiments were performed at least six times. Each symbol represents the analysis of one experiment. The lines above the bar graphs represent the significance of the standard deviations (mean; n = 10 for LAT p-Y132; n = 7 for PLC-γ1 p-Y783). For LAT p-Y132 statistical analysis: **P = 0.0098 (WT vs D at 0.06 μg/ml); *P = 0.0305 (WT vs D at 0.13 μg/ml); *P = 0.0121 (WT vs D at 0.25 μg/ml); *P = 0.0359 (WT vs D at 0.5 μg/ml); **P = 0.0036 (WT vs E at 0.06 μg/ml); *P = 0.0420 (WT vs E at 0.13 μg/ml); **P = 0.0022 (WT vs E at 0.5 μg/ml); ns = not significant: P > 0.9999 (WT vs D at 0 μg/ml); P = 0.8161 (WT vs E at 0 μg/ml); P = 0.2517 (WT vs E at 0.06 μg/ml). For PLC-γ1 p-Y783 statistical analysis: **P = 0.00206 (WT vs D at 0.13 μg/ml); ***P = 0.0005 (WT vs D at 0.25 μg/ml); *P = 0.0305 (WT vs D at 0.5 μg/ml); *P = 0.0483 (WT vs E at 0.25 μg/ml); ***P = 0.0026 (WT vs E at 0.5 μg/ml); ns = not significant: P > 0.9999 (WT vs D at 0 μg/ml); P > 0.9999 (WT vs E at 0 μg/ml); P = 0.2593 (WT vs D at 0.06 μg/ml); P > 0.9999 (WT vs E at 0.06 μg/ml); P = 0.2843 (WT vs E at 0.13 μg/ml); One-way ANOVA test.
