Skip to main content
Fig. 7 | Cell & Bioscience

Fig. 7

From: ISG15 suppresses ovulation and female fertility by ISGylating ADAMTS1

Fig. 7

ADAMTS1 degradation is mediated by ISGylation-proteasome pathway. A Western blot analysis of cumulus expansion-linked and ovulation-related genes including Ptgs2, Has2, Runx2, Tnfaip6, Ptx3, Areg, Ereg in mGCs silencing Adamts1. B, C Quantitative RT-PCR (B) analysis and western blot (C) analysis of Adamts1 and Isg15 in ovaries from PMSG/hCG stimulated wild-type mice. D Western blot analysis of ADAMTS1 in mGCs transfected with 4 μg, 8 μg or 12 μg of FLAG-ISG15 plasmid, followed by cycloheximide (CHX) treatment for the indicated duration. E ISGylation level of ADAMTS1 in mGCs transfected with 4 μg, 8 μg or 12 μg of FLAG-ISG15 plasmid. Cell lysates were immunoprecipitated with anti-HA beads. F Western blot analysis of ADAMTS1 in mGCs treated with proteasome inhibitor or autophagy inhibitor. mGCs were transfected with HA-ADAMTS1, FLAG-ISG15, FLAG-UBA7, FLAG-UBE2L6, and FLAG-HERC6. Thirty-two hours after transfection, mGCs were treated with 20 μM MG132 (proteasome inhibitor) or 5 mM 3-MA (autophagy inhibitor) for 10 h. G Immunoprecipitation assays of ISGylation of ADAMTS1 in mGCs treated with MG132. mGCs were transfected with FLAG-ISG15, HA-ADAMTS1, FLAG-UBA7, FLAG-UBE2L6, FLAG-HERC6 and 20 μM MG132. Cell lysates were immunoprecipitated with anti-HA beads. H Western blot analysis of endogenous and exogenous ADAMTS1 protein in mGCs transfected with si-Isg15, HA-ADAMTS1 plasmid or not, and MG132. I ISGylation level of endogenous ADAMTS1 in mGCs transfected with si-Isg15 and MG132. J ISGylation level of exogenous ADAMTS1 in mGCs transfected with si-Isg15, HA-ADAMTS1 plasmid and MG132. K Immunoprecipitation assays of ISGylated ADAMTS1 in mGCs treated with proteasomes. Purified ISGylated ADAMTS1 was incubated with 15 μM 20S or 26S proteasomes and analyzed by western blot. L Immunoprecipitation assays of ISGylation and ubiquitination in mGCs transfected with FLAG-USP18 plasmid and MG132

Back to article page