Quantification of PAR Accumulation in Mtz-Treated rho:YFP-NTR Larvae

To further evaluate Parp-dependent signaling downstream of Mtz treatments in rho:YFP-NTR larvae, PAR accumulation, a vetted measure of Parp activity, was quantified by western blot following the published method (Kam et al., 2018 (link)). Briefly, ~30 rho:YFP-NTR fish were treated ±2.5 mM Mtz at five dpf were collected at 3, 6, 12, 24, 48 hr post treatment (hpt). Larvae were homogenized and lysed using RIPA buffer (Sigma) supplemented with protease inhibitors (Roche). Protein concentrations were quantified using Pierce BCA kit (ThermoScientific) following the manufacturer’s instruction. Protein samples were separated on Tris-Glycine gel and then transferred onto nitrocellulose membranes. The membrane was blocked with 5% non-fat milk in TBST (Tris-buffered saline with 0.1% Tween 20) at room temperature for 1 hr and then blotted with the primary antibody, anti-human-PAR (1:2000, Dowson lab reagent), at 4°C overnight. After rinsing in TBST for three times, the secondary antibody, anti-Human IgG-HRP (1:10,000, Abcam) was applied for 1 hr at room temperature. Anti-beta-actin-HRP antibody (1:20,000) was used as a loading control. Immunolabeled bands were visualized by ECL substrate and quantified in Fiji. 24 hr post-Mtz PAR accumulation assays were performed in triplicate. Mann-Whitney U test was performed and a p-value of ≤0.05 used to indicate statistical significance.