Fig. 4: Ank5 directs ubiquitination and proteasomal degradation of NLRC5 in an F-box-dependent manner.

a, b HeLa cells were transfected to express myc-NLRC5 alone or together with Flag-Ank5 and treated with vehicle [−] or MG132 [+]. a Both input lysates and Flag IP complexes were analyzed by immunoblot. b Densitometric signal of immunoprecipitated NLRC5 divided by that of the corresponding Flag-Ank5 signal (n = 3 independent experiments). c, d HeLa cells were transfected with nontargeting (siNT) or Skp1-targeting (siSkp1) siRNA. The cells were then transfected to co-express myc-NLRC5 and either Flag-Ank5 or Flag-Ank5∆F-box. c Whole cell lysates were analyzed by immunoblot. (d) Densitometric quantification of the NLRC5:GAPDH signal ratio (n = 5 independent experiments). e, f HeLa cells were transfected to co-express myc-NLRC5 with indicated Flag-Ank5 proteins. e Whole cell lysates were analyzed by immunoblot. Vertical lines between bands indicate where the blot was cropped or imaged separately. f Densitometric quantification of NLRC5:GAPDH signal. (n = 6 independent experiments). g, h HeLa cells were transfected to co-express Flag-NLRC5 and either GFP-Ank5 or GFP-Ank5-F-boxAAAAA, treated with MG132, and cell sorted on GFP-positivity. Flag-NLRC5 was immunoprecipitated and the eluted complexes were analyzed by mass spectrometry. g Fragmentation spectra of the post-translationally modified K1194 [₁₁₇₉SPFLLANTLSLCPRVK₁₁₉₄] peptide. Spectra-matched b- (blue) and y-ion (red) fragments are displayed and summarized by the trypsinized peptide sequence inset above. h Peptide intensity presented as fold change (n = 2 independent experiments). Data are means ± SD. Unpaired two-tailed t-test (b, d, f) was used for data analysis. Source data are provided as Source Data file.