Fig. 5

ADAMTS1 is covalently modified by ISG15 in mGCs. A Workflow of immunoprecipitation-mass spectrometry. B Immunoprecipitation assays between ISG15 and ISG15-binding candidates (RNF213, RPS3, PRSS23). mGCs were transfected with FLAG-ISG15 and cell lysates were immunoprecipitated with anti-FLAG beads. C Colocalization analysis of endogenous ISG15 with ADAMTS1 by immunofluorescence assays in mGCs (left panel) and ovaries (right panel). Left panel: Anti-ISG15 and CY3-conjugated goat anti-rabbit IgG (H + L) antibodies were used to detect ISG15 (red); Anti-ADAMTS1 and FITC-conjugated goat anti-rabbit IgG (H + L) antibodies were used to detect ADAMTS1 (green); The nuclei were stained by DAPI (blue); Scale bar: 10 μm. Right panel: Anti-ISG15 and FITC-conjugated goat anti-rabbit IgG (H + L) antibodies were used to detect ISG15 (green); Anti-ADAMTS1 and CY3-conjugated goat anti-rabbit IgG (H + L) antibodies were used to detect ADAMTS1 (red); The nuclei were stained by DAPI (blue); Scale bar: 20 μm. D ADAMTS1 is a novel interaction protein of ISG15 using immunoprecipitation assays. mGCs were transfected with HA-ADAMTS1, FLAG-ISG15 or FLAG-ISG15-AA (ISG15 conjugation-defective), HA-ISG15, FLAG-ADAMTS1. Cell lysates were subjected to immunoprecipitation assays using anti-HA or anti-FLAG beads. E HERC6 is an E3 ligase for ADAMTS1 ISGylation. mGCs were transfected with HA-ADAMTS1, FLAG-ISG15, FLAG-UBA7, FLAG-UBE2L6, FLAG-HERC6 or FLAG-TRIM25 and then cell lysates were immunoprecipitated with anti-HA beads. F ISGylation of ADAMTS1 depends on the activity of E3 ligase HERC6. mGCs were transfected with HA-ADAMTS1, FLAG-ISG15, FLAG-HERC6-WT or FLAG-HERC6-CA (HERC6 ligase-deficient mutant). Cell lysates were immunoprecipitated with anti-HA beads. G USP18 decreases ADAMTS1 ISGylation. HA-ADAMTS1, FLAG-ISG15, FLAG-UBA7, FLAG-UBE2L6, FLAG-HERC6 were transfected into mGCs, together with 3 μg, 6 μg, 9 μg, or 12 μg of FLAG-USP18 plasmid. Cell lysates were immunoprecipitated with anti-HA beads