Extended Data Fig. 4: Characterization of the reporter for Lck activity at the TCR.

a, Flow cytometric analysis of ζ and ζ-Reporter expression on the surface of the ζ-, or ζ-Reporter-reconstituted or control MA5.8 cell lines using an anti-mCD3ε antibody. b, Immunoprecipitation (IP) and immunoblotting of surface TCRs using anti-mCD3ε (145-2C11) in ζ-, or ζ-Reporter-reconstituted or control MA5.8 cells. c, Scheme depicting the alternative ζ-Reporters designed to confirm constitutive exposure of the kinase-dead kinase domain of Lck within the reporter. The CD3ε proline rich sequence (PRS) was fused to the C-terminus of the ζ-Reporter. Different lengths of the CD3ε PRS were used, either containing 21 or 11 amino acids, resulting in ζ-Rep-εPRS21 or ζ-Rep-εPRS11. d and e, The expression of the ζ-Rep-εPRS constructs in MA5.8 cells was investigated as in b. f, PD assay using Nck SH3.1-beads and immunoblotting in unstimulated lysates of cells containing the ζ-Rep-εPRS reporters. The quantification of two independent experiments (mean values ± s.e.m.) is shown in the lower panel. Arrows indicated sequential PD to avoid saturation of the Nck(SH3.1)-beads. g, MA5.8 cells containing the ζ-Reporter were stimulated with plate-bound anti-mCD3ε antibodies (10 μg/ml) for 20 hours in the presence of DMSO, control peptide or a peptide inhibiting the catalytic activity of ZAP-70 (ZAP70inh). Cells were stained and measured by flow cytometry to determine the percent of CD69-positive cells. Mean values ± s.e.m. and statistical analysis using one-way ANOVA between treatments with control peptide or ZAP70inh are shown. ****P < 0.0001. h, Immunoprecipitation (IP) and immunoblotting of surface TCRs. MA5.8 cells containing the ζ-Reporter and knock out for ZAP-70 using the CRISPR/Cas9 technology were either left untreated (-) or stimulated (+) at 37 °C. One representative experiment is shown.