Extended Data Fig. 8: The CD3ε RK motif enhances CAR-Nck proximity.

a, Technical PLA controls in Jurkat T cells genetically disrupted for ζ (JKζKO) expressing the ζCAR. The cells were stimulated with Fc-CD19 at 37 °C for 5 min. PLA was performed with both primary antibodies (left), with only the anti-ζ primary antibody (middle) or with only the anti-Nck primary antibody (right). In all cases both secondary antibodies were used. The quantification of one representative experiment was analyzed using ANOVA test. Mean values ± s.e.m. are shown. b, Biological PLA controls in Jurkat T cells expressing surface TCR and Nck (left), Jurkat T cells genetically disrupted for ζ (JKζKO) (middle) and Jurkat T cells silenced for expression of Nck (JKshNck1/2) (right). The PLA was performed between the TCR (ζ) and Nck at 37 °C. Quantification of one representative experiment is shown. Statistical analysis was performed as in a. c, JKζKO cells were used to lentivirally express the CARs as indicated or were transduced with an empty vector (Mock). PLA between the CAR (ζ) and Nck in cells either left unstimulated (Uns) or stimulated with recombinant Fc-CD19 for 5 min. Data are representative of 3 independent experiments. Statistical analysis was performed using unpaired one-way ANOVA test. Mean values ± s.e.m. are shown. ****P < 0.0001.