Fig. 3: Ank5 decreases NLRC5, cellular MHC class I, and MHC class I surface levels. | Nature Communications

Fig. 3: Ank5 decreases NLRC5, cellular MHC class I, and MHC class I surface levels.

From: Orientia tsutsugamushi Ank5 promotes NLRC5 cytoplasmic retention and degradation to inhibit MHC class I expression

Fig. 3

a, b HeLa cells were mock transfected or transfected to express Flag-tagged Ank5 or Ank9 and treated with vehicle [-] or IFNγ [+]. a Inputs and Flag IP complexes were analyzed by immunoblot. b Densitometric quantification of NLRC5:GAPDH signal from input blots (n = 4 independent experiments). Indicators of significance between IFNγ-treated are blue. c–f HeLa cells were transfected to express indicated proteins, treated with IFNγ, and sorted on GFP positivity. c Whole cell lysates were analyzed by immunoblot. d–f Densitometric signal of NLRC5 (d), HLA-ABC (e), or β2 M (f) was divided by corresponding GAPDH signal and normalized to GFP-expressing IFNγ treated cells (black bars) (n = 3 independent experiments). g, h HeLa cells were mock transfected or transfected to co-express Flag-Ank5 or Flag-Ank9 with myc-NLRC5. g Input lysates and Flag IP complexes were analyzed by immunoblot. h Densitometric quantification of the NLRC5:GAPDH ratio from input blots normalized to mock samples (black bar) (n = 3 independent experiments). i–n HeLa (i, j, m, n) or EA.hy926 (k, l) cells were mock transfected or transfected to express indicated proteins and treated with IFNγ. (i, k) Whole cell lysates were analyzed by immunoblot. j, l Densitometric quantification of the NLRC5:GAPDH signal from input blots normalized to mock transfected cells treated with IFNγ (black bar, HeLa; red bar, EA.hy926) (n = 3 independent experiments). m, n MHC class I surface levels on GFP-positive cells were analyzed by flow cytometry. m Representative histograms of MHC class I levels normalized to mode. Unlabeled (black) samples were the control. [–] denotes vehicle control and [+] denotes IFNγ addition. n The mean fluorescence intensity of each sample was divided by that of its unlabeled control per replicate (n = 7 independent experiments). Data are means ± SD. Unpaired two-tailed t-test (b, d–f, h, j, l) and One-way ANOVA with Tukey’s post hoc test (n) were used for data analysis. Source data are provided as Source Data file.

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