Supplementary Figure 5: Time to achieve half-maximal recruitment of ZAP70 to phosphorylated ITAMs by wildtype LCK* in the presence of CD4 or CD8 co-receptors.

(a) Time to achieve half-maximal recruitment of ZAP70 to conjugate interface by WT LCK* in CD8⁺ HEK-TCR^H and in HEK-TCR^H cells after UV illumination at 5 mW/cm² (related to Fig. 7b). For this measure of LCK* reaction rate, a lower value indicates more efficient kinase activity. (b,c) Time to achieve half-maximal recruitment of ZAP70 to plasma membrane by WT LCK* in the absence or presence of CD8 (b) or CD4 (c) in HEK-TCR^H cells after UV illumination at 5 mW/cm² for 2 s (related to Fig. 7e,f). (d,e) Time to achieve half-maximal recruitment of ZAP70 to plasma membrane by WT LCK* in CD8⁺ and CD86^ExCD8^Int+ (d), or CD4⁺ and CD86^ExCD4^Int+ (e) HEK‑TCR^H cells relative to that in HEK‑TCR^H cells after UV illumination at 5 mW/cm² for 2 s (related to Fig. 7g,h). (f,g) Microscopy image quantification showing ZAP70 recruitment to plasma membrane after LCK* uncaging in the presence or absence of CD8 (f) or CD4 (g) in HEK-TCR^H cells after UV illumination at 500 mW/cm² for 2 s. Data are normalized to maximum asymptote values for each dataset. Lines show data smoothed used a moving-average filter, data points represent mean and filled areas represent s.e.m. from independent experiments (n = 3), where 4-8 cells were used in each independent experiment. (h,i) Time to achieve half-maximal recruitment of ZAP70 to plasma membrane by WT LCK* in the absence or presence of CD8 (h) or CD4 (i) in HEK-TCR^H cells after UV illumination at 500 mW/cm² for 2 s (related to Supplementary Fig. 5f,g). Data in a-e,h,i are presented as mean ± s.e.m. from independent experiments (n=4 in a-c; 3 in d,e,h,i).