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. 2019 Feb 25;38(6):e100024. doi: 10.15252/embj.2018100024

Figure EV3. The cullin neddylation defect in NEDP1 KO cells is not rescued by NEDD8 overexpression.

Figure EV3

  1. Overexpression of NEDD8 is insufficient to rescue the defect in Cullin neddylation in NEDP1 KO cells. Western blot analysis of whole‐cell lysates from HEK 293 WT, NEDP1 KO, or NEDP1 KO with transient overexpression of mature NEDD8. The overexpression of NEDD8 results in an increase in the amount of NEDD8 reactive species without a sufficient increase in the level of free NEDD8 to rescue the reduction of Cullin neddylation in NEDP1 KO cells.
  2. Quantification and graph of the mean ± SEM of the percentage neddylation of each Cullin in (A). One‐way ANOVA with Bonferroni post hoc test: n = 3, *P < 0.033, **P < 0.0021, n.s. denotes not statistically significant.
  3. Western blot analysis of the level of Cul3 substrate Nrf2 in HEK 293 WT, NEDP1 KO and CAND1 KO cells. Whole‐cell lysates were prepared from the indicated cell lines and analysed by SDS–PAGE and Western blot analysis. In addition, MLN4924 (3 μM) was added to WT cells for 4 h before harvested as a positive control for Nrf2 stabilization. Cul3 substrate Nrf2 is partially stabilized in NEDP1 KO and CAND1 KO cells.
  4. Quantification and graph of the mean ± SEM of the increase in Nrf2 levels in (C) from NEDP1 KO and CAND1 KO cells. One‐way ANOVA with Bonferroni post hoc test: n = 3, n.s. denotes no statistical significance.
  5. siRNA‐mediated knockdown of Cul3 substrate adaptor Keap1 stabilizes Nrf2 protein levels. U2OS cells were transfected with control siRNA (siCTRL) or siRNA against Keap1 (siKeap1), and 72 h post‐transfection, cells were lysed and processed for Western blot analysis.
  6. Nrf2 stabilization results in the induction of transcription of the Nrf2 response gene NQO1. U2OS cells were transfected with siRNA as in (D), and 48–96 h later, RNA was harvested from cells, reversed transcribed to cDNA and analysed by qPCR for Nrf2 and NQO1 expression. The graph represents mean ± SEM. Unpaired Student's t‐test: n = 6, **P = 0.0013.