Precooled ripa lysate

Brain samples or cultured cells were sonicated in precooled RIPA lysate (Beyotime) for 1 min, and 12,000 g was taken to centrifuge for 10 min at 4°C, and the supernatant was stored at −80°C. Protein concentration in the sample was determined with the BCA Protein Assay Kit (Beyotime). The extracted proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF (Millipore, Bedford, MA, USA). The membrane was sealed in a TBST buffer containing 5% skimmed milk for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-mouse-iba-1 (sc-32725, 1 : 1000), anti-mouse-TLR4 (sc-16240, 1 : 1000), anti-mouse-p-NF-κB (sc-136548, 1 : 1000), anti-mouse-β-actin (sc-47778, 1 : 1000), anti-mouse-iNOS (BD-610329, 1 : 1000), and anti-mouse- Arg-1 (sc-F0915, 1 : 1000). The membrane was then washed with TBST and incubated with horseradish peroxidase-labeled goat anti-mouse IgG (Biyuntian, A0216, 1 : 5000) and secondary antibody at room temperature for 1 h. The protein bands were detected with an enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA) and quantified with Image Lab software (Bio-Rad).

The cells were sonicated for 1 minute in the precooled RIPA lysate (Beyotime) and centrifuged at 12,000 g for 10 minutes at 4°C and the supernatant was stored at −80°C. The protein concentration in the sample was determined with BCA Protein Assay Kit (Beyotime). Electrophoretic separation was performed for the extracted protein with 10% SDS-polyacrylamide gel and the protein was transferred to PVDF (Millipore, Bedford, MA, USA). The membrane was sealed in TBST buffer containing 5% skim milk for 1 hour and then incubated overnight at 4°C with the following primary antibodies: anti-mouse-p-tlr4 (Santa Cruz, 1 : 1000), anti-mouse TLR4 (CST, 1 : 1000), anti-mouse-p-nf-κ B (Santa Cruz, 1 : 1000), anti-mouse-NF-κ B (Santa Cruz, 1 : 1000), anti-mouse-casepse3 (abcam,1 : 1000), anti-mouse-Bax (abcam,1 : 1000), anti-mouse-Bcl-2 (abcam,1 : 1000), and anti-mouse-GAPDH (Santa Cruz, 1 : 1000). Then the membrane was washed with TBST and incubated with horse radish peroxidase labeled goat anti-rabbit IgG (Beyotime, A0216, 1 : 5000) for 1 hour at room temperature. Enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA) was used to detect protein bands, and Image Lab software (Bio-Rad) was used to quantify protein bands.