Induction of IL-2 secretion by CD28 and RHOA mutants requires p300 acetyltransferase activity. (A) Luciferase reporter assays monitoring the activity of NFAT in cell lines silenced for VAV1 (small interfering RNA [siRNA] VAV1) or CTRL cells (siRNA CTRL [siCTRL], scrambled), costimulated with anti-CD3, anti-CD28, and crosslinker at 2 μg/mL for 5 hours and 30 minutes. Data are represented as mean ± SEM from 5 independent experiments conducted in quadruplicates. Significant differences in activation activity were determined using a 2-way ANOVA with Tukey multiple comparison test (∗P ≤ .05). Representative western blot of VAV1 knockdown by siRNA. Cells not used for luciferase assay were pooled according to siCTRL or siVAV1, and proteins were extracted and subjected to western blot. VAV1 signal was reduced between 24% and 29% depending on the experiment. It should be noted that only 15% to 20% of Jurkat cells are usually transduced by plasmids and siRNA by electroporation. (B) In vitro secretion of IL-2 upon stimulation with anti-CD3, anti-CD28, and crosslinker at 2 μg/mL for 24 hours with or without the specific p300 acetyltransferase inhibitor A-485 at 5 μM. Cells were pretreated with 5 μM of A-485 for 48 hours. Data are represented as mean ± SEM from 6 independent experiments conducted in quadruplicates. Significant differences in activation activity were determined using a 2-way ANOVA with Tukey multiple comparison test (∗∗P ≤ .01). Statistics not shown are indicated in supplemental Table 4.