Fig 1: Analysis of the Slp76 signalosome as a function of TCR ligand affinity.a, Dot plot showing the time-resolved interaction stoichiometry of proteins significantly associated with Slp76OST in unstimulated (Unstim.) or stimulated conditions. T cells were stimulated with N4, T4 or G4 under equal TCR occupancy conditions as described in Fig. 1a. Dots are colored-coded according to the enrichment FDR value, and only conditions with FDR values below 0.1 are represented. Proteins were considered significantly associated when presenting an enrichment FDR = 0.05 and a fold change =10 in at least one experimental condition. The interaction stoichiometry of each interacting protein was normalized by the maximal value across all experimental conditions. Proteins were ordered by hierarchical clustering based on their row-wise normalized stoichiometry. b, Immunoblot analysis of affinity-purified Slp76 molecules from OT-I Slp76OST cells that have been left unstimulated (t = 0 s) or stimulated with N4, T4 or G4 as described in Fig. 1a for the indicated times and probed with antibody to phosphorylated tyrosine (p-Tyr) or anti-SLP76. Phospho-molecules corresponding to Slp76, Lat and Cd6 are indicated by arrows. c, Immunoblot analysis of total protein lysates from OT-I cells left unstimulated (t = 0 s) or stimulated with N4, T4 or G4 as described in Fig. 1a for the indicated times and probed with a phospho-specific antibody recognizing the phosphorylated residues Y136, Y195 and Y235 in Lat. Data in b and c are representative of three independent experiments. Molecular weights are indicated on the left. d, Representation of log2-transformed fold changes of the phosphorylation intensity relative to the average intensity in unstimulated controls (t = 0 s) for the phosphorylation of the Y195 residue of Lat in the global phosphoproteome dataset. Error bars represent s.d. across biological replicates (n = 6 for N4 and unstimulated, n = 3 for T4 and G4).Source data