Las4000 mini digital imager

To detect PAPR1 activity, 30 ng of recombinant human PARP1 (Biovision, USA) was incubated with 1 µg of sonicated salmon sperm DNA (BioDynamics Laboratory Inc. Japan) and 10 mM NAD, NGD, or NHD in 30 µL of reaction buffer (20 mM Tris-HCl, pH7.4, and 10 mM MgCl₂) for 10 minutes. The reaction was terminated by adding SDS-PAGE sample buffer followed by boiling for 5 minutes at 95 °C. The samples were subjected to Western blotting with anti-PARP1 N-terminal antibody (Active Motif, USA). Chemiluminescent signals were detected by LAS 4000 Mini digital imager (GE healthcare, USA).

Cells were lysed with RIPA buffer (150 mM NaCl, 1.0% NP-40, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, and 50 mM Tris–HCl, pH 8.0) and lysates were subjected to western blotting. Antibodies used for western blotting include Nampt (Bethyl, Cat# A300-372A, Dilution 1:1000), PAR (TREVIGEN, Cat# 4336-BPC-100, Dilution 1:1000), β-actin (Wako, 017-24551, Dilution 1:500), Histone H3 (Cell signaling, Cat# 4499, Dilution 1:2000), and Histone H3K9Ac (Cell Signaling, Cat# 9649, Dilution 1:2000). Anti-mouse Nmnat1 rabbit polyclonal antibody (Dilution 1:1000) was raised against the synthetic peptide corresponding to mouse Nmnat1 residues 130–146. HRP-conjugated secondary antibodies were obtained from Millipore. PVDF membranes (Millipore) were used for blotting. Signals were detected and quantified using an LAS4000 mini digital imager (GE Health Care) and ImageQuant TL (GE Health Care), respectively.

Whole tissue lysates were prepared from WT mice. Before harvesting tissues, mice were systemically perfused with D-PBS(-) (Nacalai) via inferior vena cava (IVC) to eliminate the contamination of blood. Harvested whole tissues were grinded by Multi Beads Shocker (Yasui Kikai) with RIPA buffer (150 mM NaCl, 1.0% NP-40, 1mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, and 50 mM Tris-HCl, pH 8.0), and were subjected to Western blotting. Antibodies used for Western blotting experiments included anti-GAPDH (Sigma), anti-CoxIV (Cell Signaling) and anti-Grp75 (Abcam). Anti-mouse Nmnat3 rat monoclonal antibody (clone R88) was described previously [22 (link)]. HRP-conjugated secondary antibodies were obtained from Millipore. PVDF membrane (Millipore) was used for blotting and signals were detected by LAS4000 mini digital imager (GE Health Care).