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

Figure 6. NEDD8 trimers are acetylated to increase binding to the second zinc finger of PARP‐1.

Figure 6

  1. 2D gel electrophoresis of lysate from WT cells followed by Western blot analysis with α‐NEDD8 antibody indicates free NEDD8 migrates near its predicted isoelectric point (pI) of 6.59.
  2. 2D gel electrophoresis of pulldowns from NEDP1 KO lysate with GST‐Zn1 + 2 followed by Western blot analysis with α‐NEDD8 antibody. Some of the 25‐kDa NEDD8 band migrates to the same isoelectric point as unconjugated NEDD8 (denoted by white triangle), indicating the species is most likely an unanchored NEDD8 trimer. However, the majority of NEDD8 trimer migrates to multiple spots of lower pI (indicated in the brackets), which suggests that NEDD8 trimers undergo a post‐translational modification that either adds a negative charge or blocks a positive charge on the NEDD8 trimer.
  3. Overexpression of HDAC1 or HDAC2 decreases PARP‐1‐NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP‐1 (GFP‐Zn1 + 2). Cells were also co‐transfected, as indicated, with empty FLAG vector, HDAC1‐FLAG, or HDAC2‐FLAG. Twenty‐four hours post‐transfection, cells were harvested and immunoprecipitation was performed on the lysates with GFP‐Trap. Immunoprecipitated proteins were resolved by SDS–PAGE followed by Western blot analysis with the indicated antibodies.
  4. HDAC inhibition increases PARP‐1‐NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP‐1 (GFP‐Zn1 + 2). Twenty hours later, cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 h. Cells were harvested, and immunoprecipitation was performed on the lysates with GFP‐Trap. Immunoprecipitated proteins were resolved by SDS–PAGE followed by Western blot analysis with the indicated antibodies.
  5. HDAC inhibition increases PARP‐1‐NEDD8 trimer binding. NEDP1 KO U2OS cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 h. Cells were harvested, and lysates were incubated with recombinant Zn1‐GFP or Zn2‐GFP (25 nM) for 1 h followed by immunoprecipitation with GFP‐Trap. Immunoprecipitated proteins were resolved by SDS–PAGE followed by Western blot analysis with the indicated antibodies.
  6. Overexpression of HDAC1 or HDAC2 in NEDP1 KO cells can rescue the induction of PAR polymer following H2O2 treatment. U2OS WT or NEDP1 KO cells were transfected with the indicated FLAG vectors. Forty‐eight hours post‐transfection, cells were treated with H2O2 (600 μM) for the indicated amount of time and harvested directly in sample loading buffer. Lysates were resolved by SDS–PAGE and processed for Western blot analysis with the indicated antibodies.