Fig 1: ISGylation and Influenza B NS1 Inhibit ISG15 Secretion(A) HEK293T cells were transfected in transwell plates with plasmids expressing FLAG-ISG15, ISG15 E1 and E2 enzymes, and Herc5 (WT or C-to-A mutant), as indicated. NK-92 cells were in the upper chamber, ± IL-12, as indicated. Cell culture supernatants were monitored for IFN-? secretion by ELISA 48 h post-transfection.(B) HEK293T cells were transfected with FLAG-ISG15 with or without the ISG15 E1 and E2 enzymes and Herc5 (WT or CA mutant). Cell culture supernatants and total cell lysates were collected after 48 h. Total cell lysates (left panel) were analyzed by immunoblot with anti-FLAG antibody; supernatants were subjected to anti-ISG15 immunoprecipitation followed by an anti-FLAG immunoblot (right panel).(C) HEK293T cells were transfected in transwell plates with plasmids expressing FLAG-ISG15 and either WT NS1B or the W36A/Q37A mutant (M), as indicated. Cells were co-cultured with NK-92 cells ± IL-12, as indicated, and cell culture supernatants were monitored for IFN-? secretion by ELISA.(D) Western blots of HEK293T cells transfected with FLAG-ISG15 and either WT GFP-NS1B or the W36A/Q37A mutant (M). Total cell lysates were analyzed by immunoblotting with anti-GFP antibody (top panel) to detect GFP-NS1B or anti-FLAG antibody (middle panel) to detect FLAG-ISG15. Cell culture supernatants were subjected to an anti-ISG15 immunoprecipitation followed by an anti-FLAG immunoblot to detect secreted ISG15 (lower panel).
Fig 2: Determinants Required for ISG15 Secretion(A) FLAG-tagged ISG15 variants were transfected into HEK293T cells in the lower level of a transwell plate, with NK-92 cells in the upper chamber (± IL-12, as indicated), and secreted IFN-? was measured by ELISA. L72A, S83A, and L85F (red bars) were deficient in inducing IFN-? secretion from the NK-92 cells. Ordinary one-way ANOVA was performed between each treatment condition and the IL-12 alone condition. *p = 0.01, **p = 0.001, ***p = 0.0001, ****p < 0.0001. ns, non-significant changes.(B) Purified ISG15 protein and the indicated ISG15 variants were added directly to the cell culture media of NK-92 cells (± IL-12), and IFN-? secretion was monitored by ELISA. ISG15 variants L72A, S83A, and L85F (red bars) and wild-type (WT) ISG15 stimulated IFN-? secretion, whereas the Y96L/Q102D (LFA-1 binding mutant) did not.(C) HEK293T cells were transfected with FLAG-tagged WT ISG15 or the indicated variant. Cell culture supernatants were monitored by anti-FLAG immunoprecipitation and immunoblotting with anti-FLAG antibody. Only WT FLAG-ISG15 was detected in the supernatant of transfected cells. See Figure S1 for uncropped blots.(D) Space-filling and backbone structures of ISG15 (PDB: 1Z2M) showing the residues required for binding to LFA-1 in blue and the residues required for ISG15 secretion in red.
Fig 3: Viral De-ISGylases (vDIGs) Enhance ISG15 Secretion(A) HEK293T cells were transfected in transwell plates with plasmids expressing FLAG-ISG15 with or without the E1, E2, and E3 enzymes, and in the absence or presence of the WT or active-site CA mutants of the indicated TAP-tagged vDIG (colored bars). NK-92 in the upper chamber and cell culture supernatants were monitored for IFN-? secretion by ELISA 48 h post-transfection.(B) HEK293T cells were transfected as indicated with FLAG-ISG15 and ISG15 E1, E2, and E3 plasmids, and WT or CA NTAP-vDIGs. Total cell lysates were analyzed by immunoblotting with anti-FLAG (top panel) to detect FLAG-ISG15 or anti-TAP (two lower panels) to detect the vDIGs. Cell culture supernatants were subjected to an anti-FLAG immunoprecipitation followed by an anti-FLAG immunoblot to detect secreted ISG15 (second panel from the top).
Fig 4: TLR Agonists and IFN-ß Stimulate ISG15-Dependent IFN-? Secretion from Multiple Cell Types(A) PBMCs (±IL-12) were treated with Pam3CSK4 or poly(I:C) in the presence and absence of anti-ISG15 antibody (I) or control IgG (C). IFN-? secretion was monitored by ELISA.(B) Mouse splenocytes from control C57B6, ISG15-/-, and CD11a-/- mice were treated with Pam3CSK4 or poly(I:C) as indicated. IFN-? secretion was monitored by ELISA.(C) Primary NK cells and NK-92 cells were treated with IFN-ß in the presence and absence of anti-ISG15 antibody (I) or control antibody (C). IFN-? was monitored by ELISA.
Fig 5: Microbial Pathogens Stimulate ISG15-Dependent IFN-? Secretion from Multiple Cell Types(A) Human PBMCs were treated with recombinant ISG15, live BCG, heat-killed M. tuberculosis, or heat-killed S. typhimurium, ± IL-12 as indicated. Where indicated, anti-ISG15 antibody (I) or control IgG antibody (C) was added to the cell culture media. IFN-? secretion was measured by IFN-? ELISA.(B) Primary NK and T cells were isolated from human blood and treated with heat-killed M. tuberculosis ± IL-12 and anti-ISG15 (I) or control antibody (C), as indicated. IFN-? secretion was measured by ELISA.(C) Splenocytes from control C57B6, ISG15-/-, and CD11a-/- mice were treated with heat-killed M. tuberculosis or heat-killed S. typhimurium ± IL-12. IFN-? secretion was monitored by ELISA.
Supplier Page from Aviva Systems Biology for ISG15 ELISA Kit (Mouse) (OKCA02472)