Fig 2: Impact of TCR ligand affinity on proximal phosphorylation events.a, Representation of log2-transformed fold changes of phosphorylation intensity relative to the average intensity in unstimulated controls (t = 0 s) for phosphorylation on tyrosine residues within ITAMs of the Cd3ζOST molecule. Phosphorylation intensities were quantified from affinity-purified Cd3ζOST samples. Discrimination scores, computed as described in Fig. 3a, are reported in parenthesis. Error bars represent s.d. across biological replicates (n = 6 for N4 and unstimulated, n = 3 for T4 and G4). b, Immunoblot analysis of total protein lysates from OT-I cells left unstimulated (t = 0 s) or stimulated with N4, T4 or G4 under equal TCR occupancy conditions as described in Fig. 1a. T cells were stimulated for the indicated times and probed with phospho-specific antibodies recognizing phosphotyrosine residues in Zap70. Anti-ZAP70 and anti-GAPDH immunoblots served as a loading control. c, Immunoblot analysis of affinity-purified Cd3ζ molecules from OT-I Cd3ζOST cells treated and probed as in b. Data are representative of three independent experiments.Source data

Fig 3: Cd3-Zap70 interaction and Cd3 phosphorylation as function of ligand affinity.Immunoblot analysis of affinity-purified Cd3ζ molecules from OT-I CD3 ζ OST cells that have been left unstimulated (t = 0 s) or stimulated with N4, T4 or G4 for the indicated times. (a) Immunoblot was probed with anti-ZAP70 and anti-CD3ζ. (b) Two different levels of occupancy have been calculated and tested according to two N4 doses (4 and 0.8 nM). Immunoblot was probed with antibodies specific for phospho-tyrosines (p-Tyr) and CD3ζ. Phospho-species of CD3 chains are annotated with arrows. Molecular weights are indicated on the left (kDa). Data are representative of three independent experiments. Source data