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

Figure EV5. NEDD8 trimers bind to the DNA‐binding domain of PARP‐1 to regulate PARP‐1‐dependent gene expression.

Figure EV5

  1. Expression of TNF‐α induced and PARP‐1‐dependent gene CXCL10 is increased in NEDP1 KO cells. WT, NEDP1 KO and CAND1 KO HEK 293 cells were plated in 12‐well plates. Twenty‐four hours later, cells were left untreated or treated with TNF‐α (10 ng/ml) for 4 h, and then, RNA was harvested. cDNA was generated from the RNA, and then, qPCR was performed with primers for IκBα, CXCL10 and 18S. The TNF‐α induced expression of IκBα or CXCL10 was compared to stable 18S transcription. Fold induction for NEDP1 KO and CAND1 KO cells was then normalized to the induction in WT for each experiment. Graphs represent mean ± SEM of the normalized induction from each replicate. ANOVA with Bonferroni post hoc test: n = 3, *P < 0.033, n.s. denotes not statistically significant.
  2. Increased expression of CXCL10 following TNF‐α stimulation in NEDP1 KO cells is rescued by re‐expression of WT NEDP1. WT or NEDP1 KO HEK 293 cells plated in 12‐well plates and transfected with empty vector or p‐CMV NEDP1. Forty‐eight hours later, cells were left untreated or treated with TNF‐α (10 ng/ml) for 4 h, and then, RNA was harvested. cDNA was generated from the RNA, and then, qPCR was performed with primers for CXCL10 and 18S. The TNF‐α induced expression of CXCL10 compared to stable 18S transcription. Fold induction for NEDP1 KO was then normalized to the induction in empty vector transfected WT cells for each experiment. Graphs represent mean ± SEM of the normalized induction from each replicate. ANOVA with Bonferroni post hoc test: n = 3, **P < 0.0021, n.s. denotes not statistically significant.
  3. GST and GST‐PARP‐1 Zn1 + 2 were purified from BL21 cells and resolved by SDS–PAGE and visualized with Coomassie stain. GST‐PARP‐1 Zn1 + 2 but not GST can enrich NEDD8 trimers from NEDP1 KO lysate.
  4. 2D gel electrophoresis of GST pulldowns from NEDP1 KO lysate followed by Western blot analysis with α‐NEDD8 antibody indicates that the NEDD8 trimers in Fig 6B are specific to GST‐PARP‐1 Zn1 + Zn2 pulldown only.
  5. Diagram which depicts the evolutionary conservation of lysines residues (blue text) in mature NEDD8 from Caenorhabditis elegans to Homo sapiens.