The E3 Ubiquitin Ligase SYVN1 Plays an Antiapoptotic Role in Polycystic Ovary Syndrome by Regulating Mitochondrial Fission

Figure 4

SYVN1 controls ubiquitination and proteasomal degradation of Drp1 in KGN cells. (a) KGN cells were either transfected or not transfected with empty or SYVN1 overexpression plasmids, and western blotting was performed to examine protein expression of SYVN1 and Drp1 (). and . One-way ANOVA with Tukey’s multiple comparison test. SYVN1: , . , ; Drp1: , . , . (b) Western blot analysis of the expression of SYVN1 and Drp1 in KGN cells transfected with control siRNA or SYVN1 siRNA (). and . Two-tailed unpaired Student’s -test. SYVN1: , , ; Drp1: , , . (c) SYVN1 binding to Drp1 was detected by immunoprecipitation. (d) Western blotting was used to detect the effect of SYVN1 on Drp1 ubiquitin modification. (e) Western blot analysis of the expression of Drp1 in KGN cells transfected with either empty or SYVN1, in the presence or absence of the proteasome inhibitor MG132 (). All blots used β-actin as loading controls. . Two-way ANOVA with Sidak’s multiple comparisons test. , . (f) A cycloheximide (CHX) chase assay was performed to assess the half-life of Drp1. KGN cells were transfected with empty or SYVN1 for 24 h. Cells were then treated with CHX (100 μg/ml) for the indicated hours, and western blotting was performed. Relative Drp1 protein levels in KGN cells were quantified and plotted with the length (days) of CHX treatment on the axis. The data are expressed as .

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