Monoclonal anti parp1

Whole-cell lysates were prepared using RIPA lysis buffer (Millipore, 20–188) and protein concentration was detected using a BCA protein assay kit (Thermo Scientific). A total of 150 μg of cell lysates was mixed with anti-RIP3 (2 μg, GeneTex, USA) or MagSi-protein A/G beads (50 μL, MagnaMedics, Netherlands) at 4°C overnight. The beads were then collected using a magnet for 2 min, washed with PBST washing buffer three times, and subjected to elution with 40 μL of 1x SDS loading buffer; the samples were incubated at 95°C for 10 min. Protein was separated using SDS-PAGE and transferred to 0.2 μM PVDF membranes (Bio-Rad, USA). Blots were then probed with anti-RIP1, anti-RIP 3, anti-MLKL (GeneTex), monoclonal anti-AIF (Cell Signaling), polyclonal anti-COX4 (GeneTex), polyclonal anti-PCNA (GeneTex), monoclonal anti-PARP1, monoclonal anti-PAR (Cell Signaling), anti-phospho-AMPKα (Thr172), anti-AMPKα, anti-phospho-p70 S6 kinase (Thr389), anti-p70 S6 kinase (Cell Signaling, USA), MitoProfile Total OXPHOS rodent antibody cocktail (MitoSciences, OR), and mouse anti-GAPDH (Abcam, USA). Signals were obtained using an enhanced chemiluminescence kit (Millipore) and densitometry was performed using Fusion-Capt software (Vilber Lourmat, Fusion FX7, France).