(A) Representative Western blotting of total and phosphorylated Zap-70, p65, and ERK1/2 proteins before and at 30 min and 12 hours after the addition of anti-CD19 beads to the indicated CAR T cells at a bead–to–T cell ratio of 5:1. The anti-CD19 CAR T cells used expressed CARs with no signaling domain (Δζ), the CD3ζ chain intracellular domain alone (ζ), the CD28 and CD3ζ chain intracellular domains (28ζ), or the 4-1BB and CD3ζ chain intracellular domains (BBζ). (B to E) Quantification of the relative abundances of phosphorylated Zap-70 (B), p65 (C), ERK1 (D), and ERK2 (E) normalized to their respective total proteins from the experiments represented in (A). P values were determined by two-way ANOVA with Tukey’s multiple comparisons test of data from three donors analyzed in three independent experiments. FC, fold change; TP, time point; NTD, non-transduced.