LAT regulates phosphorylation of ZAP70 and CD3Z.

(A) The comparison of CD3ζ and ZAP70 phosphopeptides kinetics in CL20 and JCaM2.5 cell lines. (B) SILAC labeling strategy for direct comparison of peptide-specific phosphorylation in JCaM.2.5 and JCam2.5-LAT cell lines. Cell lines grown in SILAC media (Arginine: R and Lysine: K) were activated (or not) for the indicated time points. Total protein extract were equally mixed and subjected to phosphoproteomics analysis (for details see Methods and Fig. 1S). Anti-pY detection of the cell lysates was used to control the activation. Anti-ZAP70 antibodies were used to control the loading. The arrow at 18 KDa shows a band that probably corresponds to CD3ζ. L, M and H stand for Light, Medium and Heavy amino acids combinations. (C) S-shape graphic showing log2-transformed ratios for tyrosine phosphorylated peptides signals in different experimental conditions: M/L (Blue diamonds; peptide signal from 0.5 min activated JCam2.5/resting JCam2.5LAT), H/L (light-red squares; JCaM2.5LAT activated/JCaM2.5 resting), H/M (Pistachio-green triangles; JCaM2.5LAT activated/JCaM2.5 activated). (D) Similar to C except that only activated cell lines were confronted, as indicated. The graphic shows log2-transformed H/L ratio (black diamonds; JCaM2.5LAT activated/JCaM2.5 activated). (E) LAT-dependent phosphorylation of ZAP70 was tested as indicated. (F) Quantitation of the immunoblots in (E). This file contains: additional information on the analysis of global dynamics of TCR-induced phosphorylation; methods to evaluate experimental error and define activation threshold; supporting references.(A) 对比CL20与JCaM2.5细胞系中CD3ζ与ZAP70磷酸化肽的动力学特性。(B) 用于直接比对JCaM.2.5与JCam2.5-LAT细胞系中肽段特异性磷酸化水平的稳定同位素氨基酸标记培养（SILAC）策略。将在SILAC培养基（精氨酸：R与赖氨酸：K）中培养的细胞系按指定时间点进行激活（或不激活处理）。将总蛋白提取物等比例混合后进行磷酸化蛋白质组学分析（详细步骤参见方法部分与补充图1S）。采用抗磷酸化酪氨酸（pY）检测对细胞激活状态进行质控，使用抗ZAP70抗体对上样量进行质控。18 kDa处的条带可能对应CD3ζ。L、M、H分别代表轻、中、重氨基酸标记组合。(C) S型曲线图展示了不同实验条件下酪氨酸磷酸化肽段信号的log₂转换比值：M/L（蓝色菱形，对应0.5分钟激活的JCam2.5与静息JCam2.5LAT的肽段信号）；H/L（浅红色方形，对应JCaM2.5LAT激活组与JCaM2.5静息组）；H/M（开心果绿色三角形，对应JCaM2.5LAT激活组与JCaM2.5激活组）。(D) 与(C)类似，但仅按指定方式比对激活状态的细胞系，该图展示了log₂转换后的H/L比值（黑色菱形，对应JCaM2.5LAT激活组与JCaM2.5激活组）。(E) 按指定方式检测了T细胞活化连接蛋白（LAT）依赖性的ZAP70磷酸化水平。(F) 对(E)中的免疫印迹结果进行定量分析。本文件包含以下内容：T细胞受体（TCR）诱导的磷酸化全局动力学分析相关补充信息；评估实验误差与确定激活阈值的方法；以及辅助参考文献。