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.