Functional assessment of patient variants. (A) Cumulative luminescence of cells cotransfected with NADPH oxidase components (gp91^phox, p67^phox, p47^phox) and specific RAC2 mutants (30-minute time course). Unstimulated cells (basal) or after addition of 1 μM PMA (PMA). Bottom row of lower set shows expanded y-axis to detect minor activation levels. (B) Summary bar graphs from integrated kinetics of unstimulated (left) or PMA-stimulated (right) superoxide production normalized to WT basal. Dashed and solid lines correspond to WT basal and WT PMA-stimulated levels, respectively. Bars shows average ± standard of the mean (SEM), n = 3-5 independent experiments. (C) Summary of RAC2 protein stability quantified by Western blot densitometry. Graph shows RAC2/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels, normalized to WT, and expressed as mean ± SEM, n = 3 to 5. (D) Summary of RAC2-PAK1 binding expressed as bound/total mutant RAC2 normalized to WT bound/total, determined by densitometry of Western blots, displayed as mean ± SEM, n = 3-5 independent experiments. (E) Summary of pAKT levels determined by densitometry of Western blots. pAKT levels were normalized to RAC2 expression to control for protein stability and GAPDH for cell loading. Values are mean ± SEM, n = 3 to 4 independent experiments. In all plots, significance determined by Kruskal-Wallis test using 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli comparing RAC2 variants with WT RAC2; ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001.