Extended Data Fig. 1: NEDD8^R74K knock-in (KI) and NEDP1 knockout (KO) strategy for sNUSP.

a, Representative immunoblotting of total cell lysates of Nedp1^WT and Nedp1^KO CRISPR HEK293 cell clones (KO #1: sgRNA 1 + 4; KO #2: sgRNA 2 + 5), with anti-NEDD8, anti-NEDP1, anti-tubulin, and anti-actin antibodies. b, Rescue of neddylation patterns in Nedp1^KO cells by re-expression of NEDP1^WT. Nedp1^WT and Nedp1^KO HEK293 cell clones were transiently transfected with pcDNA3.1^EMPTY, NEDP1^WT-HA, NEDP1^C163A-HA, or CSN5^WT-FLAG constructs, and treated with DMSO or 2 μM MLN4924 for 24 h, as indicated. Representative immunoblotting of total cell lysates with anti-NEDD8, anti-NEDP1, anti-tubulin, anti-actin, and anti-GAPDH antibodies. c, Analysis of neddylation and ubiquitination patterns of CRISPR Nedd8^{WT or R74K} Nedp1^{WT or KO} HEK293 cell clones. Representative immunoblotting of total cell lysates from indicated cell clones, treated with DMSO or 3 μM MLN4924 for 24 h, with anti-NEDD8, anti-ubiquitin (FK2), anti-tubulin, anti-actin, and anti-GAPDH antibodies. Uncropped blot images are provided as source data. Experiments were repeated at least three times with similar results. Molecular mass is indicated in kilodaltons.

Source data