Fig. 7: NLRC5 K-to-R mutants are resistant to O. tsutsugamushi-induced degradation and antagonize Orientia mediated reduction of MHC class I surface levels. | Nature Communications

Fig. 7: NLRC5 K-to-R mutants are resistant to O. tsutsugamushi-induced degradation and antagonize Orientia mediated reduction of MHC class I surface levels.

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

Fig. 7

a–c HeLa cells expressing indicated proteins were treated with IFNγ or vehicle, sorted on GFP-positivity, and resolved into cytoplasmic [C] and nuclear [N] fractions. a Samples were analyzed by immunoblot. b, c The densitometric signal of cytoplasmic (b) or nuclear (c) NLRC5 was divided by that of Hsp90 or lamin A/C, respectively (n = 3 independent experiments). d, e HeLa cells were transfected to co-express indicated myc-NLRC5 proteins and Flag-tagged Ank5. d Inputs and Flag IP complexes were analyzed by immunoblot. e Densitometric quantification of myc:GAPDH signal from inputs (n = 3 independent experiments). f, g HeLa cells were transfected to express indicated GFP-NLRC5 proteins, mock infected [U] or infected with O. tsutsugamushi [I], and treated with IFNγ at 2 h p.i. (f) At 24 h p.i., whole cell lysates were immunoblotted. g Densitometric quantification of GFP:GAPDH signal from inputs was normalized to matched uninfected controls (n = 7 independent experiments). h HeLa cells were transfected to co-express Flag-BAP or Flag-Ank5 with indicated GFP-NLRC5 proteins and treated with IFNγ or vehicle (n = 4 independent experiments). MHC class I surface levels of GFP-positive cells were analyzed by flow cytometry. Indicators of statistical significance between pairs of untreated (black bars) and IFNγ-treated (white bars) samples are colored black, between untreated samples compared to BAP [–] are colored blue, and between IFNγ-treated samples compared to BAP [+] are colored red. i U and I HeLa cells were transfected to express indicated GFP-NLRC5 proteins and treated with IFNγ or vehicle for 24 h (n = 3 or 4 independent experiments). At 48 h p.i., samples were collected and assessed for MHC-I surface levels by flow cytometry. Fold change mean fluorescence intensity (MFI) was calculated by subtracting the MFI of each vehicle control-treated sample from that of its IFNγ-treated counterpart and then dividing that by its vehicle control. Data are means ± SD. One-way ANOVA with Dunnett’s post hoc test (b, c, h) and Unpaired two-tailed t-test (e, g, h, i) were used for data analysis. Source data are provided as Source Data file.

Back to article page