Poly adp ribose polymerase 1 parp1

The protein samples were separated using 8%, 10%, and 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to polyvinylidene difluoride membranes (Cat# ISEQ. 00010, LOT# R6PA4145H; Merck Millipore, USA). The membranes were blocked with 5% skim milk for 3 h at 37°C and were incubated for 14 h at 4°C with the following diluted primary antibodies: pyruvate kinase (PK; 1:1,000; Wanlei, China), uncoupling protein 1 (UCP1; 1:1,500; Wanlei), succinate dehydrogenase (SDH; 1:500; Bioss), pyruvate dehydrogenase complex (PDHX; 1:500; Affinity, USA), lactate dehydrogenase (LDH; 1:1,000; Wanlei), poly ADP-ribose polymerase 1 (PARP1; 1:500; Proteintech, China), Cas8 (1:1,000; CST, USA), nuclear factor kappa B (NF-κB; 1:500; Wanlei), interleukin-1β (IL-1β; 1:1,000; Wanlei), and TNF-α (1:500; Wanlei). After washing thrice for 15 min each with phosphate-buffered saline with Tween 20, the membranes were incubated for 2 h at 37°C with peroxidase-conjugated secondary antibodies against rabbit IgG (Cat# sc-2357, RRID: AB_628497; Santa Cruz Biotechnology, Argentina). After washing three times by PBST for 15 min each again, the bound antibodies were visualized through chemiluminescence by using the ECL-plus reagent (GE Healthcare, UK). The GAPDH content was analyzed as the loading control by using a rabbit polyclonal antibody.