Extended Data Fig. 2: The SH3 domain of Lck binds to the CD3ε cytoplasmic tail.

a, Sequence of the murine CD3-derived peptides used in the study. b, A pull-down (PD) assay using glutathione beads bound to GST (-) or to GST-fusion proteins containing either the Lck SH2 domain (SH2) or both Lck SH2 and SH3 domains (SH2-SH3) was performed. These beads were incubated with the indicated biotinylated peptides as shown in (a) (PP refers to doubly phosphorylated ITAMs). Immunoblotting was performed using streptavidin-HRPO and anti-GST antibodies. c, Flow cytometric analysis in Jurkat T cells retrovirally transduced with scrambled or CD3ε-specific shRNAs before and after FACS sorting of GFP⁺ cells. The new Jurkat T cell variants were named H8 (scrambled shRNA) and H8εsh (CD3ε-specific shRNA). d, Immunoprecipitation of surface TCRs from H8 and H8εsh cells and immunoblot analysis (a long and a short exposure for CD3ε are shown). e, Flow cytometric analysis of murine (mCD3ε) and endogenous human CD3ε (hCD3ε) expression in H8εsh cells reconstituted with murine CD3εWT, CD3εPRSΔ or CD3εK76T using specific antibodies (145-2C11 and UCHT1, respectively). Murine 2B4 and human Jurkat T cells were used as controls. f, Cells as in e were left untreated (-) or stimulated for 5 min at 37 °C with 5 μg/ml anti-mCD3ε antibody. A PD using Nck SH3.1-beads detects TCRs that have switched to the active conformation and thereby exposed the PRS in CD3ε.