p.Met41Val produces less UBA1b as compared with other patient variants. (A) p.Met41Val supports less UBA1b translation than p.Met41Thr and p.Met41Leu. HEK293T cells were transfected with C-terminally FLAG-tagged WT UBA1 and indicated VEXAS variants, lysed, subjected to anti-FLAG immunoprecipitation and analyzed by anti-UBA1a/b/c or anti-UBA1a/b immunoblotting. Shorter and longer exposures of the respective immunoblots are shown. (B) Quantification of the experiment shown in panel A. Error bars denote standard error of the mean, n = 3 technical replicates, *P < .05; **P < .01, ordinary 1-way ANOVA. (C) p.Met41Val carriers express less UBA1b than p.Met41Thr and p.Met41Leu carriers. CD3-depleted peripheral blood mononuclear cells (PBMCs) of healthy volunteers (C1, C2) and VEXAS patients with similar VAFs were subjected to anti-UBA1a/b/c or anti-UBA1a/b immunoblotting. (D) Quantification of anti-UBA1a/b immunoblots of CD3-depleted PBMCs or CD14⁺ cells of healthy volunteers (control) or VEXAS patients (p.Met41Val/Thr/Leu). Error bars denote standard deviation, n = 8 (4 biological replicates of independently processed patient samples per genotype with 2 technical replicates each; technical replicates of each biological replicate are depicted in the same color and symbol), *P < .05, ordinary 1-way ANOVA.