Fig. 6: Ank5 inhibits MHC class I expression in an AR4-dependent but F-box-independent manner.
a–i HeLa cells were transfected to express indicated proteins, treated with IFNγ or vehicle control, and sorted on GFP-positivity. a–c Total RNA was collected and RT-qPCR was performed using gene-specific primers. Relative expression of NLRC5-to-GAPDH (a), HLA-A-to-GAPDH (b), and β2M-to-GAPDH (c) was determined using the 2-∆∆CT method in which conditional values were normalized to those values of GFP-expressing cells treated with IFNγ (black bars). (n = 5 independent experiments). d–g Whole cell lysates were collected from sorted cells and analyzed by immunoblot (d). Vertical lines between bands indicate where the blot was cropped or imaged separately. e–g The densitometric signal of NLRC5 (e), HLA-ABC (f), or β2 M (g) was divided by the corresponding GAPDH densitometric signal and normalized to GFP-expressing cells treated with IFNγ (black bars) (n = 3 independent experiments). h, i Surface levels of MHC class I from sorted cells were analyzed by flow cytometry. h Representative histograms of MHC class I levels normalized to mode. Unlabeled (black) samples served as the control. [–] denotes vehicle control and [+] denotes IFNγ addition. i The mean fluorescence intensity of each sample was divided by that of its unlabeled control per replicate (n = 4 independent experiments). Data are means ± SD. One-way ANOVA with Dunnett’s post hoc test (a–c), Unpaired two-tailed t-test (e–g), and One-way ANOVA with Tukey’s post hoc test (i) were used for data analysis. Source data are provided as Source Data file.
