Ac nf b

For western blot analysis, equal protein amounts per lane were separated by 10% SDS-polyacrylamide gels and transferred to a polyvinylidenedifluoride membrane. The membrane was blocked in 5% skim milk for 2 h at room temperature and incubated overnight at 4 °C with primary antibodies against SIRT1 (cat# sc-15404, 1:200, Santa Cruz, CA, USA), Iba-1 (cat# sc-98468, 1:200, Santa Cruz, CA, USA), ac-FoxO1 (cat# sc-49437, 1:200, Santa Cruz, CA, USA), ac-p53 (cat# 2570, 1:400, Cell Signaling, MA, USA), ac-NF-кB (cat# 12629 S, 1:400, Cell Signaling), Bcl2 (cat# sc-492, 1:200, Santa Cruz, CA, USA), Bax (cat# sc-493, 1:200, Santa Cruz, CA, USA), caspase-3 (cat# 9661, 1:400, Cell Signaling), histone 3 (cat# BS7416, 1:2000, Bioworld, MN, USA) and β-actin (cat# AP0060, 1:5000, Bioworld) in tris-buffered saline with Tween-20 containing 5% skim milk. After the membrane was washed, it was incubated with horseradish peroxidase-conjugated IgG for 2 h at room temperature. The blotted protein bands were visualized by enhanced chemiluminescence western blot detection reagents (Thermo Scientific, Kalamazoo, MI, USA) and were exposed to X-ray film. The quantification of band density was performed using the UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA).

Protein extracts were separated by 10% or 15% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio‐Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% non‐fat milk for 2 hours, and then they were incubated overnight at 4°C with SIRT1 (1:200, Santa Cruz Biotechnology), Bcl‐2 (1:200, Santa Cruz Biotechnology), Cleaved caspase‐3 (1:1000, Cell Signaling Technology, Danvers, MA, USA), Bax (1:200, Santa Cruz Biotechnology), caspase‐3 (1:400, Cell Signaling Technology), ac‐NF‐кB (1:400, Cell Signaling Technology), IL‐6 (1:200, Santa Cruz Biotechnology), TNF‐α (1:200, Santa Cruz Biotechnology) and β‐actin (1:5000, Bioworld Technology, St. Louis Park, MN, USA) in blocking buffer. After three washes with TBST for 15 minutes each, the membranes were subsequently incubated in secondary antibody conjugated with horseradish peroxidase (HRP) (1:1000, Bioworld Technology) at room temperature for 2 hours. The protein bands were exposed to the Tanon‐5200 Chemiluminescent Imaging System, and strip grey levels were quantified by software (version 4.62; Bio‐Rad Laboratories).