Fig. 4.

CD45 increases the unbinding rate of ZAP70 from ITAMs beyond the ZAP70 off-rate. (A) Example sensogram of the CD45-accelerated ZAP70 unbinding assay in SPR. ZAP70 (500 nM) was first injected over a surface of the ITAM3–phosphorylated CD3ζ cytoplasmic domain and allowed to reach steady state before a mixture of ZAP70 (500 nM) and CD45 (indicated concentration) was injected, and finally, ZAP70 (500 nM) was injected. (B) The ZAP70 and CD45 coinjection phase for multiple concentrations of CD45 demonstrating a concentration-dependent acceleration in the loss of ZAP70 binding. (C) The fitted observed unbinding rate over [CD45] (results from three experiments conducted on different days are shown). The ZAP70 off-rate is shown as a dashed line with ± SEM shaded in gray. (D) The regulated off-rate calculated over [CD45]. Binding of ZAP70 to this full-length Avi-CD3ζ ITAM3 was the same as on the shorter ITAM3 peptide (SI Appendix, Fig. S4).