Imagej program

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).

Total proteins were extracted using radioimmunoprecipitation assay buffer (RIPA) with 1% PMSF (Solarbio, Beijing, China) after transfection for 48 h or induction for six days after transfection. The extracted protein was mixed with protein loading buffer (denaturation) at a ratio of 4:1 and denatured at 100 °C for 10 min. Then, protein was subjected to 10% polyacrylamide gel electrophoresis at 80 V for 30 min and 120 V for 90 min and transferred to a 150 V ice bath for 40 min. The skim milk powder was blocked for 1 h, and the primary antibody was incubated overnight. The antibodies were then rinsed with PBS-Tween three times for 5 min each. The fluorescent secondary antibody was incubated in the dark for 2 h and washed with PBST three times for 5 min each. The primary antibodies used were against cyclin D1 (1:1000, bs-20596R, Bioss), proliferating cell nuclear antigen (PCNA; 1:1000, bs-0754R), peroxisome proliferator-activated receptor γ (PPARγ; 1:1000, bs-4590R), CCAAT/enhancer-binding protein alpha (C/EBPα; 1:1000, bs-1630R), phospho-beta-arrestin (ARRB1; 1:1000, bs-3048R), and GAPDH (1:5000, bs-2188R). The secondary antibody used was goat anti-rabbit IgG H&L/AP (1:5000, bs-0295G-AP). Finally, we present the results of Western blotting using Image Studio Lite ver. 5.2 (LI-COR Inc., Lincoln, NE, USA) and quantified with the ImageJ program (Bio-Rad, Hercules, CA, USA).

Cells treated with different concentrations of LIG, SENA and SENI for 8 hours were subject to Western blot assay. Proteins were extracted from whole cell lysates and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, then transferred to a polyvinylidene fluoride (PVDF) membrane. The following primary antibodies were used: rabbit anti-P-gp (1:1000; Abcam, Cambridge, MA, USA) and mouse anti-β-actin (1:2000; Abcam). Membranes were then incubated with the horseradish peroxidase-conjugated secondary anti-bodies (1:5000; Abcam). The transferred proteins were incubated with enhanced chemiluminescence (ECL) substrate solution and visualized with the Image J program (Bio-Rad, Richmond, CA, USA). The relative levels were quantified densitometrically by using the Quantity One software (Bio-Rad Laboratories) and calculated according to the reference bands of β-actin.