Fig 2: Prognostic value of LCP2 in lung adenocarcinoma (LUAD) patients. (a) Kaplan–Meier curves showing the overall survival of LUAD patients with high and low LCP2 mRNA expression. (b) Kaplan–Meier curves showing the overall survival of LUAD patients with high and low LCP2 protein expression. *P < 0.05.

Fig 3: Comparative analysis of the TCR-triggered SLP-76 interactomes of human primary CD4+ T cells in the presence or absence of dasatinib. (A) Immunoblot analysis of equal amounts of proteins from total lysates of SLP-76OST and control human CD4+ T cells that were expanded for AP-MS and either left unstimulated (0 s) or stimulated for 30 or 120 s with anti-CD3 and anti-CD28 and probed with antibody to phosphorylated tyrosine (anti–p-Tyr), anti–SLP-76, or anti-ß actin (loading control). Before stimulation, T cells were incubated for 45 min at 37°C with either dasatinib (100 nM) or vehicle alone (DMSO) as specified. Dasatinib (100 nM) and DMSO were also kept during all the subsequent steps until cell lysis. Data are representative of at least two independent experiments. (B) Dot plot showing the interaction stoichiometry of SLP-76 with its high-confidence TCR regulated preys over the course of TCR-CD28 stimulation and in the presence of dasatinib or vehicle (DMSO). The stoichiometry of the constitutive SLP-76–GRAP2 interaction is also shown. For each SLP-76–prey interaction, the interaction stoichiometry has been row-normalized to its maximum value observed over the course of TCR-CD28 stimulation (see normalized stoichiometry [Norm. Stoichio.] key). Also shown is the P value corresponding to the enrichment in OST-tagged versus untagged AP-MS samples (see P value key). (C) Plot comparing the enrichment (shown as normalized intensity, see Materials and methods) of GRAP2, VAV1, 14-3-3 ?, and CD6 in AP-MS samples from SLP-76OST (OST) and control (WT) human CD4+ T cells over the course of TCR-CD28 stimulation in the presence of dasatinib or vehicle (DMSO). ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, P = 0.05, where P corresponds to a Welch’s two-sided t test P value.

Fig 4: 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

Fig 5: Identification of the functional domains that mediate the interaction between RAGE and SLP76.a Schematic diagram of the protein domains of RAGE and SLP76. The specific domains/motifs of RAGE (upper panel) and SLP76 (lower panel) are shown in distinct colors. Different truncations or mutants of RAGE and SLP76 were constructed and are displayed in the upper and lower panels, respectively. b Mapping the region of RAGE that binds with SLP76. Flag-tagged full-length SLP76 and HA-tagged WT RAGE or its deletion mutants were overexpressed in HEK293 cells. After treatment with or without AGEs (100 µg/ml) for 15 min, cells were collected for lysis. Immunoprecipitation was performed with a specific antibody against the HA tag to detect the interaction between RAGE and SLP76 (n = 3). **P < 0.01, ***P < 0.001 versus untreated cells, ####P < 0.0001 versus AGE-treated cells cotransfected with full-length RAGE and SLP76. c Identification of the functional domain of SLP76 that interacts with RAGE. HA-tagged full-length RAGE and Flag-tagged WT SLP76 or its mutants were overexpressed in HEK293 cells. Immunoprecipitation with a specific antibody against the HA tag was performed to identify the domain of SLP76 interacting with RAGE (n = 3). *P < 0.05, ****P < 0.0001 versus cells coexpressing full-length RAGE and SLP76. d The SAM domain mediates the in vivo interaction between RAGE and SLP76. Plasmids expressing ECFP-tagged RAGE and EYFP-tagged SLP76 or SLP76(?SAM) were coexpressed in HEK293 cells. FRET assays were performed to verify the binding activity of the SAM domain of SLP76 with RAGE (n = 5). e Quantification of the in vivo interaction of RAGE and SLP76 (n = 5). The FRET efficiency was further quantitated by the formula Net FRET (N-FRET) = FRET signal - (a*YFP signal) - (b*CFP signal) as described in the “Methods”. The results for each group are shown. ****P < 0.0001 versus cells cotransfected with full-length RAGE and SLP76. Data are presented as mean ± SD in (b), (c), and (e). Statistics were performed using one-way ANOVA (b, c, and e) followed by Tukey post hoc. Scale bar: 10 µm. Source data are provided as a Source data file.