RHOA mutations in AITL and TFH-like PTCL. (A) Overview of the RHOA protein structure, showing G17V (42 AITL, 6 TFH-like PTCL) and the novel K18N (3 AITL) variants that target the highly conserved GTP/GDP binding site of RHOA. (B) Protein blot analysis of GTP-bound RHOA-Myc in rhotekin pull-down assay from HEK293T cells expressing indicated RHOA constructs. IP, immunoprecipitation. Representative of 6 independent experiments. (C) SRE (serum-responsive element) luciferase reporter assay monitoring the activity of RHOA K18N mutant, compared with WT, G14V, or G17V mutants, previously characterized as activating and dominant-negative, respectively. Cells were stimulated (light gray) or not (dark gray) with fetal bovine serum for 6 hours. Data are represented as mean ± standard error of the mean (SEM) from 4 independent experiments. Significant differences in activation activity were determined using 2-way analysis of variance (ANOVA) with repeated measurement (**P ≤ .01; ***P ≤ .001 compared with WT). Representative western blot from a luciferase assay experiment. Ectopic myc-tagged RHOA expression is revealed by anti-Myc. Anti-Actin blotting serves as loading control.