Fig. 6: Neddylation deficiency elevates NIK which contributes to hepatocyte damage.
a Western blot in whole liver extracts of AAV-GFP (GFP) and AAV-Cre (Cre) mice at D21 and D24 post-virus injections (n = 6 per group). b Immunoblot analyses in mouse Nae1f/f primary hepatocytes (PH) 48 h after infection with pAd-GFP and pAd-Cre (n = 3 per group). Arrow indicates the expected murine NIK. c Eighteen hours after transfection with NIK-HA, HepG2 cells were treated with vehicle (Veh) or 1.0 µM MLN for 12 h before 20 µg/ml CHX treatment for indicated periods. d Western blot and quantifications in HepG2 cells treated with Veh, 1.0 µM MLN with or without 5 µM B022 for 48 h. 20 ng/mL TNFα was added in the last 24 h. Cleaved caspase-3 and NIK signaling were quantified by normalizing to cells receiving MLN and TNFα (n = 5 per group). Veh (black), B022 (blue). Multiple unpaired t-tests with the Holm-Sidak method. e, f qRT-PCR analyses in (e) HepG2 cells (n = 3 per group) and (f) wild-type primary hepatocytes (n = 4 per group) treated with vehicle (Veh), 1.0 µM MLN with or without 5 µM B022 for 48 h. In e, f, Veh (black), MLN (red), MLN + B022 (blue). One-way ANOVA followed by Tukey’s multiple comparisons test. g–i Male Nae1f/f mice injected with AAV-Cre received Veh or B022 via daily i.p injection from D10 post-virus injection. AAV-GFP mice were also included as a control. g, h Western blot of indicated proteins and quantifications in liver extracts from D24 AAV-GFP, AAV-Cre mice receiving Veh or B022 (n = 3 per group). Data in h were normalized to AAV-Cre mice receiving Veh. Multiple unpaired t-tests with the Holm-Sidak method. Veh (black), B022 (red). i Relative mRNA levels in livers from D24 AAV-GFP, AAV-Cre receiving Veh or B022 mice (GFP: black, n = 6; Cre+Veh: red, n = 6; Cre+B022: blue, n = 3 in duplicates). Two-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05; **P < 0.005; ns: not significant. All quantitative data were presented as mean ± SEM. Source data are provided as a Source Data file.
