Figure 2. NEURL4 enzymatic activity is... | Rockefeller University Press

Figure 2.

$NEURL4 enzymatic activity is essential for mitochondrial PARylation. (A and B) Validation of NEURL4 KO cell lines generated by CRISPR/Cas9 genome editing of HeLa cells using two independent gRNA. WB of whole cell extracts (A) or fractionated cytosolic (Cyto), mitochondrial (Mito), and nuclear (Nuc) extracts (B). Rescue is performed by transient transfection with 200 ng full-length NEURL4 expressing vector. Efficiency of sub-cellular fractionation is confirmed by WB for β-tubulin, mtHSP70, and HDAC2. (C) WB anti-PAR on mitochondrial protein extracts showing that most mitochondrial PAR production is lost in untreated HeLa cells upon NEURL4 deletion. (D) WB anti-PAR on mitochondrial protein extracts from HeLa cells showing that loss of ADP-ribosylation in the absence of NEURL4 can be rescued reintroducing either full-length NEURL4 or the Ct domain alone (by transient transfection with 2 μg each). (E) NEURL4 enzymatic activity is inhibited by the generic PARP inhibitor BGP-15 (10 µg/ml) but not by PARP1/ARTD1 inhibitor olaparib (1 µM). (F) In vitro PARylation assays showing NEURL4 Ct domain ability to produce poly-ADP-ribosylation chains (wells 4, 5, and 6) compared with the catalytic mutant (well 7). FL, full length; tot, total extracts.$

NEURL4 enzymatic activity is essential for mitochondrial PARylation. (A and B) Validation of NEURL4 KO cell lines generated by CRISPR/Cas9 genome editing of HeLa cells using two independent gRNA. WB of whole cell extracts (A) or fractionated cytosolic (Cyto), mitochondrial (Mito), and nuclear (Nuc) extracts (B). Rescue is performed by transient transfection with 200 ng full-length NEURL4 expressing vector. Efficiency of sub-cellular fractionation is confirmed by WB for β-tubulin, mtHSP70, and HDAC2. (C) WB anti-PAR on mitochondrial protein extracts showing that most mitochondrial PAR production is lost in untreated HeLa cells upon NEURL4 deletion. (D) WB anti-PAR on mitochondrial protein extracts from HeLa cells showing that loss of ADP-ribosylation in the absence of NEURL4 can be rescued reintroducing either full-length NEURL4 or the Ct domain alone (by transient transfection with 2 μg each). (E) NEURL4 enzymatic activity is inhibited by the generic PARP inhibitor BGP-15 (10 µg/ml) but not by PARP1/ARTD1 inhibitor olaparib (1 µM). (F) In vitro PARylation assays showing NEURL4 Ct domain ability to produce poly-ADP-ribosylation chains (wells 4, 5, and 6) compared with the catalytic mutant (well 7). FL, full length; tot, total extracts.

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