FIGURE 2.

IL23Rα protective variants R381Q, G149R, and V362I display reduction in IL23R signaling. A, HeLa cells were co-transfected with plasmids carrying IL23Rα or variants and IL12Rβ1. After washing with PBS to remove media and overnight growth in serum-free medium, transfected cells were incubated with or without IL23 (15 ng/ml) for 30 min. Cell lysates were subjected to SDS-PAGE followed by Western blotting with antibodies specific to phospho-STAT3 (pSTAT3) and STAT3. Representative blots from three independent experiments are shown here. B, corresponding densitometry of the levels of pSTAT3 in IL23Rα and IL23Rα variants obtained from Western blots above. The signal of pSTAT3 detected on Western blot was measured as pixels using ImageJ 1.46r (National Institutes of Health) and were further normalized to levels of total STAT3. Data and S.D. are representative of three independent experiments. Statistical significance calculated by ANOVA is denoted by the asterisk (*), where p < 0.01 is compared with IL23Rα; however, R381Q was significantly different from V362I, with a p < 0.05. C, HeLa cells were co-transfected with plasmids carrying STAT4, IL23Rα or variants, and IL12Rβ1. As stated for STAT3 experiments (A), the cell lysates were subjected to SDS-PAGE followed by Western blotting with antibodies specific to phospho-STAT4 (pSTAT4) and STAT4. D, corresponding densitometry of the levels of pSTAT4 in IL23Rα and IL23Rα variants obtained from Western blots of C. The signal of pSTAT4 detected on Western blot was measured as pixels using ImageJ 1.46r (National Institutes of Health) and were further normalized to levels of total STAT4. Data and S.D. are representative of three independent experiments. Statistical significance calculated by ANOVA is denoted by the asterisk (*), where p < 0.01 is compared with IL23Rα; in addition, R381Q was significantly different from G149R and V362I with a p < 0.01. EV denotes empty vector.