Fig. 5: Ank5 AR4 is critical for binding NLRC5, the NLRC5 LRR is essential for Ank5-mediated degradation, and Ank5-F-boxAAAAA competitively antagonizes O. tsutsugamushi mediated NLRC5 degradation. | Nature Communications

Fig. 5: Ank5 AR4 is critical for binding NLRC5, the NLRC5 LRR is essential for Ank5-mediated degradation, and Ank5-F-boxAAAAA competitively antagonizes O. tsutsugamushi mediated NLRC5 degradation.

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

Fig. 5

a–d HeLa cells were transfected to co-express myc-NLRC5 and indicated Flag-tagged Ank5 proteins. a, c Input lysates and Flag IP complexes were analyzed by immunoblot. b, d Densitometric signal of immunoprecipitated NLRC5 was divided by the corresponding Flag-Ank5 protein signal and multiplied by 100 to determine percent interaction (n = 3 independent experiments). e, f Schematic of wt NLRC5 (e) and isoform 3 (f). Amino acid positions at the beginning of each domain are indicated. g–j HeLa cells were transfected to co-express myc-tagged wt NLRC5 or isoform 3 with Flag-tagged Ank5 or Ank5-F-boxAAAAA (g and h) or transfected to express myc-tagged wt NLRC5 or isoform 3 and then mock infected [U] or infected with O. tsutsugamushi [I] (i, j). g, i Input lysates, Flag IP complexes, and whole cell lysates were analyzed by immunoblot. Red asterisks denote the bands of interest. h, j Densitometric quantification of the myc:GAPDH signal was normalized to Flag-Ank5-F-boxAAAAA (h) or uninfected (j) cells (black bars) (n = 3 independent experiments). k, l HeLa cells were transfected to express indicated proteins, sorted on GFP positivity, mock infected [U] or infected with O. tsutsugamushi [I], and treated with IFNγ. k Whole cell lysates were analyzed by immunoblot. l Densitometric quantification of the NLRC5:GAPDH signal (n = 3 independent experiments). Indicators of statistical significance between pairs of uninfected (black bars) and infected (white bars) samples are colored black. Indicators of statistical significance between transfected and uninfected samples compared to uninfected cells expressing GFP-Ank5 are colored blue. Vertical lines between bands indicate where the blot was cropped or imaged separately. Data are means ± SD. Unpaired two-tailed t-test (b, d, h, j, l) was used for data analysis. Source data are provided as Source Data file.

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