SENP8 inactivation leads to an increase of unneddylated CUL4A/B and depletion of free Nedd8. (A) Representative western blot analysis of SENP8-inactivated and control sgAAVS1-transduced BC-3/Cas9 cells indicates a strong depletion of unconjugated Nedd8 and an increase in Nedd8-conjugated proteins. GAPDH served as loading control. SENP8 sg1 and sg2 are 2 independent SENP8-specific sgRNAs used. (B) Representative western blot analyses of CUL4A, CUL4B, and CRBN in SENP8-inactivated BC-3/Cas9 and BCBL-1/Cas9 cells indicate an increase in unneddylated CUL4A/B and no significant change in CRBN protein levels. Neddylated (N8) and unneddylated forms of CUL4A/B are indicated by arrows. GAPDH served as loading control. (C) Quantitative analysis of the data shown in panel B over replicates. Protein levels were first normalized to corresponding GAPDH levels and then to sg AAVS1 levels (n = 2; error bars represent SEM).