β actin

Total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membrane (Millipore Sigma, MA). After blocking with 5% skim milk, membrane was incubated overnight at 4 °C with primary antibodies against Erk1/2, phospho-Erk1/2 (Thr202/Tyr204), Akt, phospho-Akt (Ser473) (Cell Signaling Technology, MA) and β-actin (Wako, Japan). Then, membrane was incubated with either anti-mouse or anti-rabbit HRP–conjugated secondary antibodies, washed and visualized with the Western Lightning Plus ECL Substrate (Perkin Elmer, MA).

BCL-2-like protein 11 (Bim) (#2933) at 1:3000, Syk (#13198) at 1:3000, Caspase 3 (#9662) at 1:100 (Cell Signaling Technology, Danvers, MA, USA), iba-1 (016-20001) at 1:1000 (WAKO, Tokyo, Japan), β-actin (sc-47778) at 1:10,000, Cytochrome C (sc-13156) at 1:3000, Glial fibrillary acidic protein (GFAP) (sc-33673) at 1:3000, Synaptosomal-associated protein, 25 kDa (SNAP25) (sc-20038) at 1:3000, synaptophysin (SYP) (sc-17750) at 1:3000, postsynaptic density protein-95 (PSD-95) (sc-71933) at 1:3000, p-NF-kB (sc-136548) at 1:1000, TLR4 (sc-293072) at 1:1000, TNFα (sc-52746) at 1:1000 (Santa Cruz, Dallas, TX, USA), iNOS (610432) at 1:3000 (BD Lifescience, Franklin Lakes, NJ, USA), COX-2 (160126) at 1:3000 (Cayman, Ann Arbor, MI, USA).

Cells were dissolved in lysis buffer, and 10 µL of each protein sample was fractionated on polyacrylamide gels (TGX™ FastCast™ Acrylamide Kit; Bio-Rad Laboratories, Hercules, CA, USA) and then electroblotted onto nitrocellulose membranes. The membranes were blocked and incubated with primary antibodies against β-actin (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) or KLF4 (Novus Biologicals, Littleton, CO, USA), and then the membranes were incubated with horseradish peroxidase-conjugated secondary antibody. The membranes were then treated with enhanced chemiluminescence detection reagents (Amersham™; Cytiva, Marlborough, MA, USA), and chemiluminescent signals were visualized as bands using a LAS 4000 mini analyzer (Cytiva).

Human fibrosarcoma cell line HT1080 and Mouse Embryonic Fibroblasts (MEFs) cells were grown in Dulbecco’s Modified Eagle Medium (DMEM), 10% heat-inactivated fetal bovine serum (FBS), and 1% penicillin–streptomycin solution, at 37 °C under a 5% CO₂ atmosphere. siRNAs were purchased from Qiagen (Hilden, Germany) (RNF4 #1: SI03228512, RNF4 #2: SI04167828). AllStars negative control siRNA (Qiagen) was used as a control. siRNAs were transfected using Lipofectamine RNAiMAX (Merck Millipore, Burlington, VT, USA), according to the manufacturer’s protocol. All reagents were obtained from commercial sources; TNF-α (Enzo Life Sciences, Farmingdale, NY, USA), 5z-7-oxozeaenol(5Z-7), BV-6, necrostatin-1 (Santa Cruz, Dallas, TX, USA), cycloheximide (Sigma, St. Louis, MO, USA). The antibodies used were against caspase-8 (Enzo Life Sciences), RNF4 (Proteintech, Rosemond, CA, USA), FLAG (Sigma), α-tubulin, p65, Fibrillarin (Santa Cruz), caspase-3, phospho-p38, total p38, phospho-JNK, total JNK, phospho-ERK, total ERK, phospho-RIPK1 (Cell signaling, Danvers, MA, USA), total RIPK1 (Becton and Dickinson, Franklin Lakes, NJ, USA), and β-actin (Wako, Tokyo, Japan).

Equal amounts of proteins were mixed with sample buffer (4% sodium dodecyl sulfate (SDS), 10% beta-mercaptoethanol, and 20% glycerol in 0.125 M Tris, pH 6.8) containing bromophenol blue and boiled for 5 min. The samples were loaded and separated via 10% SDS-polyacrylamide gel electrophoresis (PAGE). Proteins separated via SDS-PAGE were transferred to polyvinylidene difluoride membranes (Immobilon-P transfer membrane) and blocked for 1 h with blocking buffer (5% non-fat milk in tris-buffered saline containing 0.5% Tween-20 (TBST) for 1 h. Next, the membranes were incubated overnight at 4 °C with primary antibodies for anti-cleaved caspase-3 (1:500; Cell Signaling Technology), anti-4-HNE (1:500; ab46545, Abcam), and β-actin (1:10000; no. 017–24573, Wako Pure Chemical Industries Ltd., Tokyo, Japan). Membranes were washed three times with Tween/PBS for 15 min, and then incubated with anti-rabbit immunoglobulin G (IgG) (1:1000; Cell Signaling Technology) for 1 h. Finally, membranes were washed with Tween/PBS and treated with ECL-western blot detecting reagent (Amersham Biosciences Corp., Piscataway, NJ) for visualization. β-actin was used as an internal control.

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.