FIGURE 8.

Quantitative proteomics revealed that Fbxw24 knockout altered the level of various proteins. (A and B) Fbxw24 knockout substantially reduced pan‐ubiquitination levels in PND 21 ovaries. (C) TMT‐labeled quantitative proteomics was performed to comprehensively investigate the effects of Fbxw24 knockout on mouse ovaries at the protein level. Peptides from two repeats of WT or Fbxw24‐KO ovaries were isobaric‐mass tagged by TMT6‐126, 127, 128, and 129, respectively, and analyzed by LC‐MS. (D) Heat map of 82 differentially expressed proteins (DEPs) at a 1.2‐fold threshold (Fbxw24‐KO / WT > 1.2 or < 0.833). (E) Among the 82 DEPs, 60.98% (50 out of 82) were up‐regulated, and 39.02% (32 out of 82) were down‐regulated. (F) By string 2.0, among the 82 DEPs, 60.98% (50 out of 82) connect with each other. (G) KEGG and protein interaction analysis show that six ubiquitination and degradation‐related proteins are in the centre of the interaction network (green dot‐line circle), and around them, there are eight groups of proteins; 14 proteins are involved in immunity, infection, and inflammation processes and consist of the largest group (red dot‐line circle). In particular, there are two separate groups: one group containing Cyp11a1, Cyp17a1, and Hsd3b1, all of which are involved in steroidogenesis (pink dot‐line rectangle); and the other contains Hist1h1b, Hist1h1c, and Hist1h1e, which are all involved in chromatin modulation (blue dot‐line rectangle). (H and I) Western blot verified that Fbxw24 knockout increased STAT1 protein level. (J) Co‐IP and western blot showed that FBXW24 interacted with STAT1. (K and L) Western blot verified that Fbxw24 knockout increased DTX3L protein level. (M) Co‐IP and western blot showed that FBXW24 interacted with DTX3L. Actin was used as a loading control. *, p < 0.05. AU, arbitrary unit