ADA2 protein expression and secretion in the homogenous and heterozygous state of variants F355L, T360A, and N370K on a denaturing gel. (A and E) Immunoblot of the whole-cell lysate and supernatants of HEK293T cells transfected with ADA2 variants F355L in the homozygous state, together with the WT ADA2 or with ADA2 variant Y453C in the carrier state, or HEK293T cells transfected with ADA2 variants T360A and N370K in the homozygous state or together with WT ADA2, respectively. Cells and supernatant were collected 48 h after transfection. The image shown represents two and three independent experiments, respectively. Loading control: B-actin. (B and F) Quantification of ADA2 protein expression in the whole-cell lysate of HEK293T cells transfected with WT ADA2 or ADA2 variants F355L, T360A, and N370K in homozygous conditions. Bar graphs represent the percentage of ADA2 protein expression relative to WT100% ADA2. (C and G) Quantification of ADA2 secretion in the supernatant of HEK293T cells transfected with WT ADA2 or ADA2 variants F355L, T360A, and N370K in homozygous conditions. Bar graphs represent the percentage of ADA2 protein expression relative to WT100% ADA2. (D and H) Quantification of ADA2 secretion of cotransfected HEK293T cells of ADA2 variants F355L, T360A, and N370K together with WT in heterozygous conditions. Bar graphs represent the percentage of ADA2 secretion relative to WT50% ADA2. (B–G) Each bar represents the mean ± SD from two to three independent experiments, respectively. EV, empty vector. Source data are available for this figure: SourceData FS1.