Fig 2: LFA-1 cross-linking needed for pY-171 and FAK auto-phosphorylation.(a) MgCl2/EDTA induced the adhesion of T cells to ICAM-1 on plates. Left panels: images of T cells bound to ICAM-Fc (left: untreated controls; right: MgCl2/EDTA treated cells stained with DAP1 and TRITC-phalloidin). Right panel: histogram showing MgCl2/EDTA induced increases in T-cell binding to ICAM1-Fc (n=3). (b) Antibody cross-linking is needed to induce pY-171 and FAK/PYK2 auto-phosphorylation. Jurkat cells were either incubated with soluble anti-LFA-1 Fab’ or Fab’ plus MgCl2/EDTA or full sized anti-LFA-1 for various times followed by blotting with anti-pY171- LAT, anti-LAT, anti-pY-397-FAK, anti-FAK or anti-pY-402-PYK2 and anti-PYK2. Assay was performed in the absence of ICAM1 on plates (n=3). (c) Mono-valent ICAM1 failed to induce pY-171 and FAK/PYK2 auto-phosphorylation. Jurkat cells were either incubated with soluble ICAM1-Fc or ICAM1-Fc plus MgCl2/EDTA or bivalent anti-LFA-1 for various times followed by blotting with anti-pY171- LAT, anti-LAT, anti-pY-402 PYK2 and anti-PYK2. Assay was performed in the absence of ICAM1 on plates (n=3).

Fig 3: LFA-1 induced SKAP1-LAT and reduced LAT-SLP-76 complexes.(a) In situ proximity analysis shows anti-LFA-1 induced SKAP1-LAT proximity. Murine DC27.10T-cells were ligated with anti-CD3 and/or LFA-1 followed by in situ proximity analysis for SLP-76 and LAT (upper panels) or SKAP1 and LAT (lower panels) (n=4). (b) LFA-1 cross-linking increases SKAP1-LAT micro-cluster colocalization. Jurkat T cells transfected with mCherry-LAT and GFP-SKAP1 were stimulated on anti-CD3 or ICAM1/CD3 coated cover slips. Micro-cluster formation was monitored by confocal microscopy. Upper panel: resting; middle panel: anti-CD3; lower panel anti-CD3/ICAM1. Scale bar corresponds to 10 µM. (n=3) (c) SKAP1 co-precipitates LAT and vice versa in response to anti-LFA-1. DC27.10 T cells were ligated with anti-CD3 and/or anti-LFA-1 for 5 min followed by membrane preparation and precipitation with anti-SKAP1 and blotting with anti-SKAP1, anti-LAT and anti-SLP-76 (upper panels). Conversely, cells were separated into cytosol (lanes 3,4) and membranes (lanes 5,6) and precipitated with anti-LAT followed by blotting with anti-LAT or anti-SLP-76 (n=3). (d) ICAM1 binding to LFA-1 increases the binding of SKAP1 to LAT. DC27.10T-cells were ligated with ICAM1-Fc alone on plates (2 µg ml-1) for various times followed by anti-SKAP1 precipitation and blotting with either anti-LAT, anti-SLP-76 or anti-GADs (n=3) (e) Anti-LFA-1 induced LAT-SKAP1 in human peripheral primary T-cells. Human resting T-cells ligated with plate bound ICAM1-Fc (2 µg ml-1) or ICAM1-Fc plus soluble anti-CD3 (1 µg ml-1) for various times followed by anti-SKAP1 precipitation and blotting with anti-SKAP1, anti-LAT, anti-GADS or anti-SLP-76 (n=3).

Fig 4: TCR signaling in WT and WASP-/- cells. Related to Fig 3 A TCR engagement-induced phosphorylation of early signaling molecules Zap70, SLP76, and LAT. The points in the plots represent values obtained from individual cells normalized to the mean of “WT” values.B, CColocalization of foci (derived from Phalloidin—Alexa 568 images) with phospho-Zap70 (B) and with phospho-PLC?1 (C) is substantially reduced in late (15') synapses of WT T cells. P values n.s. > 0.05; ***< 0.001; **= 0.01, measured using Mann–Whitney test. Scale bars, 5 µm.

Fig 5: GRB2 binds via its SH3 domains to SKAP1.(a) GRB2 binds to SKAP1. 293T cells were transfected with GFP-GRB2, FLAG-SKAP1 or both, followed by anti-Flag precipitation and blotting with anti-GFP or anti-Flag. Lanes 1–3: cell lysates; lanes 4–6: anti-Flag precipitations (n=3). (b) GRB2 SH3-1 and 2 domains bind to SKAP1. 293T cells were transfected with FLAG-SKAP1, followed by detergent solubilization of cells and GST pull-down with versions of GST-tagged GRB-2 constructs and blotting with anti-Flag or anti-GST (n=3). (c) GRB2 SH3 domains bind to the SK region of SKAP1. 293T cells were transfected with GFP-tagged GRB2 followed by detergent solubilization of cells, and GST pull-down with various versions of GST-SKAP1 (GST-SKAP1-WT1; GST-SKAP1-N-PH-SK; GST-SKAP1-N-PH; GST-SKAP1-PH; GST-SKAP1-SH3; GST-SKAP1-SK) and blotting with anti-GFP or anti-GST (n=3). (d) GST-SKAP1 co-precipitates LAT, GRB-2 and ADAP from Jurkat T-cells. Jurkat cells were ligated with anti-LFA-1 for 5 min. followed by lysis of cells, pull down with GST, GST-SKAP1 WT or GST-SKAP SH3 domain followed by blotting with anti- PLC?1, anti-LAT, anti-GRB-2, anti-ADAP and anti-SLP-76 and anti-GST. GST-SKAP WT pulled down LAT, GRB-2, ADAP, but neither PLC?1 nor SLP-76 (n=3).