Protein Extraction and Western Blot Analysis

Whole cell protein extraction, cytosolic protein extraction, and nuclear protein extraction were extracted according to previous studies (Pan et al., 2014 (link); Zhang et al., 2016a (link)). Equal protein amounts were separated by polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked in 5% skim milk for 2 h at room temperature and then incubated with primary antibodies against caspase-3 (1:400, cat #9661, Cell Signaling), SIRT1 (1:200, cat #SC-15404, Santa Cruz), HMGB1 (1:1000, cat #3935S, Cell Signaling), TLR4 (1:200, cat #sc-30002, Santa Cruz), Myd88 (1:200, cat #sc-11356, Santa Cruz), Nf-κB p65 (1:200, cat #sc-372, Santa Cruz), β-actin (1:3000, cat #AP0060, Bioworld Technology, Minneapolis, MN, USA), and Histone H3 (1:3000, cat #BS7416, Bioworld Technology) overnight at 4°C. The membrane was incubated with horseradish peroxidase (HRP)-conjugated IgG for 2 h at room temperature. Detection was performed by enhanced chemiluminescence solution (Thermo Fisher Scientific, Waltham, MA, USA). Band density was quantified with UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA).