Characterization of the Jurkat cell lines used in this study. (A) Representative western blot from the 5 modified Jurkat cell lines. Expression of ectopic Myc-tagged CD28 and HA-tagged RHOA was revealed by anti-Myc and anti-HA, respectively. Anti-actin blotting serves as a loading control. Positions of molecular weight markers are indicated in kilodalton. (B) Representative flow cytometry analysis of the cell lines showing CD28 expression level at the plasma membrane. Expression levels of CD28 T195P were slightly higher than those of CD28 wt. (C) Relative RHOA expression measured by qPCR in all 5 cell lines. The 4 cell lines expressing exogenous RHOA showed similar levels of RHOA expression. Data are expressed as mean ± standard error of the mean (SEM) of 3 independent experiments. ∗∗∗∗P < 0.0001. (D) SRE luciferase reporter assay monitoring the activity of RHOA wt, compared with G17V mutant, which was previously characterized as dominant negative. Cells were stimulated or not with FBS for 6 hours. Data are represented as mean ± SEM from 3 independent experiments. Significant differences in activation were determined using 2-way analysis of variance (ANOVA) with Tukey multiple comparison test (∗∗∗P ≤ .001, compared with cells expressing RHOA wt). The color code used for the 5 Jurkat cell lines generated for this study is indicated at the bottom of the figure. FBS, fetal bovine serum; SRE, serum-responsive element.