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. 2020 Jan 15;117(5):2473–2483. doi: 10.1073/pnas.1910765117

Fig. 5.

Fig. 5.

ETFA and ETFB are neddylation substrates. (A) The interaction between Myc-tagged ETFs and endogenous Ubc12 was analyzed by IB after IP with an anti-Myc antibody. (B) The interaction between endogenous ETFs and endogenous Ubc12 in postnatal-day-7 liver tissues was analyzed by IB after IP with an anti-Ubc12 antibody or a control IgG antibody. HC, heavy chain; LC, light chain. (C–E) IB analysis of an entire molecular weight spectrum of ETF expression with whole-cell lysates harvested from postnatal-day-7 liver tissues (C) or BNL CL.2 cells at 0, 8, 12, or 16 h after treatment with 0.5 μM MLN4924 (D) or 96 h after infection with the indicated lentiviral vectors (E). ShRNA, short hairpin RNA. (F and G) The neddylation of endogenous ETF proteins in postnatal-day-7 liver tissues (F) or BNL CL.2 cells treated with 0.5 μM MLN4924 for 0, 8, 12, or 16 h (G) was examined by IB after IP under partially denaturing conditions. ns, nonspecific. (H) Primary hepatocytes from 8-wk-old 6 × His-FLAG-NEDD8 heterozygous KI and littermate WT mice were treated with or without 2 μM MLN4924 for 24 h, and the neddylation of endogenous ETF proteins was then analyzed by histidine pulldown. Please note that in F–H, the amount of lysate in each sample was adjusted to ensure comparable protein levels of ETFA and ETFB. (I) Twenty-four hours after transfection with 2 μg FLAG-NEDD8 and varying amounts of Myc-ETF expression constructs, cells of hepatic origin in 60-mm dishes were treated with or without 0.5 μM MLN4924 for another 24 h. The neddylation of exogenous ETF proteins was then examined by IB after IP under partially denaturing conditions. (J) The smear bands obtained in SI Appendix, Fig. S11, were subjected to mass spectrometry analysis. Possibly modified peptides are shown. (K) Possibly neddylated lysines identified by mass spectrometry analysis and adjacent evolutionarily conserved lysines were mutated to arginines. The neddylation of Myc-tagged ETFs and the corresponding 4R mutants was then examined as described in I. (L) Forty-eight hours after BNL CL.2 cells were transfected with 0.5 μg His-NEDD8 and varying amounts of GFP-ETF expression constructs in 60-mm dishes, the neddylation of exogenous ETF proteins with or without the 4R mutation was then examined by histidine pulldown. Please note that in I–L the amounts of tagged ETFA or ETFB plasmid in each sample were adjusted to ensure comparable protein levels.