Fig. 6
From: ISG15 suppresses ovulation and female fertility by ISGylating ADAMTS1
Lys309, Lys593, Lys597, and Lys602 are the covalent ISG15 conjugation sites on ADAMTS1. A ADAMTS1 contains at least 2 ISGylation sites. mGCs were transfected with HA-ADAMTS1, HA-USP18, FLAG-ISG15 or FLAG-ISG15-8KR (ISG15 Lys-less variant). Cell lysates were immunoprecipitated with anti-HA beads. B Schematic representation of full-length ADAMTS1 (FL) and its deletion mutants (F1-F5). TSP: thrombospondin type 1 repeats. C Mapping the interacting regions in ADAMTS1 with ISG15. mGCs were transfected with FLAG-ISG15, HA-ADAMTS1, the truncation mutants of ADAMTS1 (F1-F5). Cell lysates were immunoprecipitated with anti-HA beads. D Identification of ISGylation sites on ADAMTS1 F2. E Identification of ISGylation sites on ADAMTS1 F3. 3KR1: K545/549/553R. 3KR2: K593/597/602R. F Lys309, Lys593, Lys597, and Lys602 are ISGylation sites on ADAMTS1. 2KR1: K309/593R, 2KR2: K309/597R, 2KR3: K309/602R, 4KR: K309/593/597/602R. (D-F) HA-ADAMTS1 is subjected to mutagenesis for substituting the Lys residues with Arg residues. mGCs were transfected FLAG-ISG15, HA-ADAMTS1 and the indicated point mutants of ADAMTS1 (K-to-R mutants). Cell lysates were immunoprecipitated with anti-HA beads. G Western blot analysis of ovulation-related genes including Adamts1, Ptgs2, Has2, Areg, Ereg in mGCs overexpressing ADAMTS1 mutants