Enhanced chemiluminescence technique

Nuclear and cytoplasmic protein was isolated from mouse brain substantia nigra and cell using the Beyotime Nuclear and Cytoplasmic Protein Extraction Kit and RIPA buffer (Beyotime). The NanoDrop 2000 was used to quantify protein concentrations (Thermo Fischer Scientific, USA). Electrophoretically, protein extracts were separated on sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels before being transferred to a PVDF membrane (Merck Millipore, Germany). Primary antibodies were incubated at 4°C overnight after being blocked in 5% BSA in the TBST buffer for 1 h at room temperature. Anti-actin (1 : 5000), anti-LC3B (1 : 1000), anti-p62 (1 : 1000), anti-pmTOR (1 : 3000), anti-TLR2 (1 : 500), anti-TLR4 (1 : 500), anti-NF-kappa B p65 (1 : 500), and anti-histone H3 (1 : 500) primary antibodies were diluted as described. The PVDF membrane was incubated with HRP-labeled IgG antibody (1 : 10000) in TBST for 2 hours at room temperature after being washed three times in TBST for 30 minutes. Quantitative studies were performed using the ImageJ program and the enhanced chemiluminescence technique (Bio-Rad).