Fig 2: Inhibiting Csk increases the magnitude of ligand-induced TCR signaling and reduces the threshold for TCR activation.(A) Purified CskAS (AS) or wildtype (WT) CD4+ T cells stimulated for 2 min with 1 µg/ml, 5 µg/ml or 10 µg/ml anti-CD3e antibody in the presence of DMSO or 10 µM 3-IB-PP1 were analyzed by immunoblotting for the phosphorylation of the activation loop tyrosine of Lck and Fyn (Src pY416 antibody) and the inhibitory tyrosine of Lck (Lck pY505), phosphorylated ZAP-70 (ZAP70 pY319), LAT (LAT pY132) and PLC-?1 (PLC?1 pY783), as well as total actin (loading control). Data are representative of at least three independent experiments. (B) Purified total CskAS (AS) or wildtype (WT) T cells stimulated with the indicated dose of anti-CD3e antibody for 2 min in the presence of DMSO or 5 µM, 1 µM or 0.4 µM 3-IB-PP1 were analyzed for phosphorylated ERK (p-ERK) by phosphoflow. Histograms were gated on CD4+ cells. Data are representative of at least three independent experiments for AS cells and two independent experiments for WT cells.DOI: http://dx.doi.org/10.7554/eLife.08088.003

Fig 3: Proposed model for the role of FES in neutrophil phagocytosis via SYK-HS1-PLC?2 pathway activation.Representations are simplified for illustration purposes.

Fig 4: H5 ImmTAAI localizes at the target cell–T cell interface and inhibits proximal TCR signaling.(A) Schematic of the cell assay used to visualize H5 ImmTAAI localization. (B) Phase contrast and confocal fluorescence images of H5 ImmTAAI localization on ECN90 ß cell target cells cocultured with either PD-1+ or PD-1– Jurkat cells. Upper panel: phase contrast. Lower panel: fluorescence images, a white arrow indicates ImmTAAI accumulation (representative images from 3 independent studies). Total original magnification, ×100. (C) The numbers of ECN90 ß cell: Jurkat conjugates with ImmTAAI accumulation (white arrow) were counted and plotted as a percentage of the total number of cell conjugates (n = 100 conjugates for conditions using Jurkat Mel5 PD-1 cells and n = 96 for Jurkat Mel5 cells, plotted as mean data from 3 independent studies). (D) Jurkat Mel5 PD-1 cells were stimulated with Melan-A–pulsed ECN90 or ECN90 PD-L1 cells for the indicated times. Whole cell lysates for each condition and time point were resolved by SDS-PAGE, transferred to PVDF membranes, and probed by Western blotting for phospho–SLP-76 (pSLP-76), total SLP-76 (SLP-76), phospho-PLC? (pPLC?), total PLC? (PLC?), phospho–ZAP-70 (pZAP-70), and total ZAP-70 (representative blots from 3 independent experiments). (E) Jurkat Mel5 PD-1 cells were stimulated with Melan-A–pulsed ECN90 cells, in the presence or absence of H5 ImmTAAI, and Western blots were performed as above (representative blots from 3 independent experiments).

Fig 5: Zap70 tSH2 is recruited to LIC-Z-YFP clusters in reconstituted COS-7. COS-7 cells were reconstituted to co-express LIC-Z-YFP, mCherry-tagged Zap70 tSH2 and wild-type Lck GFP. (a,b) Representative images of Zap70 tSH2, LIC-Z-YFP and Lck GFP colocalization in COS-7 cells immediately fixed after 2 min irradiation with blue light (a) and corresponding Pearson coefficient analysis (b); Scale bar = 10 µm. In (b), the third column is Pearson coefficient values between LIC-Z and Lck GFP channel in COS-7 cells expressing only these two constructs. Data are mean and standard error of n = 20 cells. * p < 0.05, (one-way ANOVA with Fisher LSD post hoc test). (c,d). Representative images of the dynamic recruitment of Zap70 tSH2 to newly formed LIC-Z-YFP clusters in live COS-7 cells (c) and frame-by-frame Pearson coefficient values (d) between Zap70 tSH2 and LIC-Z-YFP channel; Scale bar = 3 µm. Each trace is one cell.