Ecl western blot detection kit

Cells were lysed on ice for 10 min in RIPA buffer (Beyotime Biotechnology). The cell lysates were then centrifuged at 10,000
g , and the supernatants were collected for western blot analysis. Equal amounts of protein were separated by SDS-PAGE and blotted onto PVDF membranes. Membranes were incubated initially with primary antibodies and subsequently with corresponding secondary antibodies for 1 h. Specific bands were visualized using an enhanced chemiluminescence (ECL) western blot detection kit (Beyotime Biotechnology).

Hepatocytes were washed twice and collected in ice-cold PBS. Total protein extracts were obtained using a reducing SDS buffer containing 50 mmol/L Tris-HCl (pH 6.8), 100 mmol/L DTT, 2% SDS, and 10% glycerol. Protein concentrations were determined for diluted samples using the Bradford procedure. Equal amounts of protein (100 μg) were separated by 6% SDS-PAGE and transferred onto membranes. The membranes were blocked in a TBS solution containing 5% nonfat dry milk and incubated with an antibody against ACCα (1:1000 dilution; Beijing Biosynthesis Biotechnology, China). A goat anti-rabbit horseradish peroxidase-conjugated IgG (1:2000; Beijing Biosynthesis Biotechnology, China) was used as the secondary antibody, and the signals were detected using an ECL western blot detection kit (Beyotime Institute of Biotechnology, China). After analysis, the membranes were blotted with ananti-α-tubulin antibody (1:1000; Beijing Biosynthesis Biotechnology, China) to normalize for protein content. The blot images were digitized using a luminescent image analyzer (LAS-1000, Fuji Photo Film). The protein bands were quantified using Quantity One software (Bio-Rad). The relative ACCα protein level was determined based on the value of ACCα expression divided by the value of α-tubulin expression. Each individual experiment was repeated three times and averaged.

The spinal cord of the lumbar (L₄-L₅) ipsilateral quadrant to the lesion was collected, dissected, and homogenized in protein lysis buffer in the presence of protease inhibitors and incubated on ice for 10 min. The samples were centrifuged at 12,000 rpm for 15 min at 4°C. The total protein content was determined in the supernatants using the Bio-Rad DC Protein Assay Kit. Equal amounts of protein were resolved by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% nonfat milk at room temperature and incubated overnight at 4°C with primary antibodies (rabbit anti-Iba1, ab178680, 1 : 1000, Abcam, USA; rabbit anti-BDNF, ab108319, 1 : 1000, Abcam, USA; rabbit anti-p-ERK, #4370, 1 : 1000, Cell Signaling Technology, USA; rabbit anti-PI3K, ab40776, 1 : 2000, Abcam, USA). Then, the membranes were incubated with a horse-radish peroxidase-conjugated secondary antibody (1 : 5000, Thermo Scientific, USA) at room temperature for 2 h. Finally, peroxidase activity was visualized using the ECL Western Blot Detection Kit (Beyotime, China). Western blots were quantitated using an image analysis system (Bio-Rad, USA). After normalization with β-actin, the data were presented as mean percentages of the ratio of total protein to their respective signal intensity levels found in the Sham group animals, indicated as 100%.