RHOA G17V and CD28 T195P synergize in promoting NFAT and AP1 transcriptional activity upon CD3/CD28 stimulation. (A) Representative western blot and densitometry quantification of PLCγ1 and VAV1 phosphorylation from cell lines stimulated with anti-CD3, anti-CD28, and crosslinker at 2 μg/mL during the indicated periods of time before protein extraction. The CD28 activating mutant, but not RHOA G17V, induced increased phosphorylation of PLCγ1 and VAV1. Total PLCγ1 and VAV1 served as controls (CTRLs) for the quantification of the phosphorylated forms. Positions of molecular weight markers are indicated in kilodalton. (B-C) Luciferase reporter assays monitoring the activities of NFAT (B) and AP1 (C) upon stimulation of cell lines either with IgG CTRL or anti-CD3, anti-CD28, and crosslinker at 2 μg/mL for 5 hour and 30 minutes. Data are represented as mean ± SEM from 4 to 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; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001). (D) In vitro secretion of IL-2 upon costimulation with anti-CD3, anti-CD28 and crosslinker at 2 μg/mL with or without a small dose of CsA (120 nM) for 24 hours. Data are represented as mean ± SEM from 4 independent experiments conducted in quadruplicates. Significant differences in activation activity were determined using a 2-way ANOVA with Tukey multiple comparison test (∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001). Statistics not shown are indicated in the Supplemental Table 3.