FIGURE 5.

Localization of IL23Rα common and protective variants in HEK293 cells. A, confocal microscopy images of cells expressing N-terminal FLAG-tagged receptors in HEK293 cells. Cells were imaged for vYFP fluorescent protein to show IL23Rα expression levels; Hoeschst (Nuc-Blue, Molecular Probes), to identify the nucleus and antibody toward FLAG tag, was used to localize IL23Rα. Overlay images (Merge) from Hoechst and anti-FLAG staining is shown to demonstrate IL23Rα localization. Scale bar, 10 μm. B, mean fluorescent intensities from the corresponding confocal images were used to quantify IL23Rα, which were normalized to the total expression of IL23Rα and its variants using levels of vYFP fluorescent protein as described under “Experimental Procedures.” Different fields of images were quantified, and S.E. values are n = 3. Statistical significance calculated by ANOVA is denoted by asterisk (*), where p < 0.01 compared with IL23Rα.R381Q was significantly different from G149R and V362I, and G149R is significantly different from V362I with a p < 0.01. C, localization of IL23Rα variant-vYFP fusions; green, IL23Rα; red, ER. Hoeschst staining was used to identify the nucleus, and cell light ER-RFP was used to identify ER. Scale bar, 10 μm. D, ratio of total fluorescence from v-YFP-tagged IL23Rα and ER-localized v-YFP-tagged IL23Rα is calculated as described under “Experimental Procedures.” On average, 10 cells were quantified for each variant, and the S.E. was calculated. Statistical significance calculated by ANOVA is denoted by the asterisk (*) compared with IL23Rα, where p < 0.05; in addition, R381Q was significantly different from G149R, with a p < 0.05, and G149R was significantly different from V362I, with a p < 0.01.