Radio immunoprecipitation assay cell lysis buffer

Spinal cord tissue homogenates or total cellular proteins were extracted using radio‐immunoprecipitation assay cell lysis buffer (Beyotime, Nanjing, Jiangsu, China). Protein concentrations were determined with the Bicinchoninic Acid Protein Assay Kit (Beyotime). Subsequently, samples underwent 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Thermo Fisher Scientific) for protein separation, followed by protein transfer onto polyvinylidene fluoride (PVDF) membranes via wet transfer (electrotransfer for 2 h in 70 V at 4°C in a cold room). The PVDF membrane was blocked in 5% skim milk‐TBS with Tween 20 (TBST) and incubated at room temperature for 1 h. Next, the membrane was probed with specific primary antibodies (Abcam, Cambridge, UK) anti‐FTO (1:1000, ab280081), anti‐HO‐1 (1:2000, ab189491), and anti‐GAPDH (1:2500, ab9485) at 4°C overnight. Following incubation and TBST washing, the membranes were treated with horseradish peroxidase‐labeled goat anti‐rabbit secondary antibody immunoglobulin G (IgG; 1:2000, ab205718, Abcam) at room temperature for 1 h. Membranes were subjected to chemiluminescence for detection, and protein band density was quantified using the Image J software (National Institutes of Health, Bethesda, Maryland, USA). GAPDH served as an internal reference, and each experiment was repeated three times.

The binding relation among ZFAS1, and miR-144-5p was validated using the RIP test kit (Millipore, MA, USA). Cells were washed with pre-cooled PBS, and then lysed on ice with an equal volume of radio-immunoprecipitation assay cell lysis buffer (Beyotime, Shanghai, China) , then centrifuged . Cell extracts were divided into two parts for inputs and co-precipitation, respectively. During co-precipitation with antibodies, 50 μL magnetic beads were resuspended in 100 μL RIP wash buffer, and incubated with  anti-Ago2 (Abcam, MA, USA) and anti-immunoglobulin G (IgG) (1:5000; 7074; Cell Signaling Technology, MA, USA), respectively. After that, the magnetic bead-antibody complex was washed and resuspended in 900 μL RIP wash buffer followed by overnight incubation at 4°C with 100 μL cell extract. The purified complex was collected, processed with protease K, and followed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) [28 (link)].