Peroxidase hrp conjugated secondary antibody

Total cellular proteins were isolated by incubating the cells with RIPA buffer containing 1% PMSF (Sigma) on ice for 30 minutes. The protein concentrations were quantified by BCA Protein Assay Kit (Beyotime). Proteins were electrophoresed by 10% SDS‐PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were then blocked with 5% skimmed milk for 2 hours at room temperature. Subsequently, membranes were incubated with the primary antibodies, including anti‐YY1 (63227, Cell Signaling Technology 1:1000) and anti‐β‐actin (3700, Cell Signaling Technology 1:1000) for overnight at 4°C. Next morning, the membrane was incubated with peroxidase (HRP)‐conjugated secondary antibody (Proteintech, 1:5000) for 1 hour at room temperature. Finally, the intensity of the bands was detected by the chemiluminescence system (Bio‐Rad) and analysed by Image Lab software.

Protein was extracted from the rat lung tissues with lysis buffer (RIPA: PMSF=100:1), and equal amounts of protein from each sample (20 μg or 40 μg) were separated by 8% or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After incubation with high-affinity anti-YTHDF1 antibody (1:1000, Proteintech), anti-YTHDF2 antibody (1:5000, Proteintech), anti-METTL3 antibody (1:1000, Abcam), anti-METTL14 antibody (1:1000, Bioss Antibodies), anti-FTO antibody (1:1000, Abcam), anti-ALKBH5 antibody (1:1000, Abcam), anti-WTAP antibody (1:5000, Proteintech), anti-VIRMA antibody (1:1000, Abcam), anti-RBM15 antibody (1:1000, Proteintech), anti-YTHDF3 antibody (1:1000, Abclonal), anti-YTHDC1 antibody (1:1000, Proteintech), anti-YTHDC2 antibody (1:1000, Abclonal), anti-IGF2BP1 antibody (1:1000, Abcam), anti-IGF2BP2 antibody (1:1000, Abcam), anti-IGF2BP3 antibody (1:1000, Abcam), and anti-β-actin antibody (1:5000, Proteintech), the membranes were then incubated with peroxidase (HRP)-conjugated secondary antibody (1:5000, Proteintech). The chemiluminescent signals were detected with a chemiluminescent HRP substrate [51 (link)]. Densitometric analysis was conducted with ImageJ software.

Cells treated with lentivirus were lysed with RIPA (Beyotime, China) buffer containing protease inhibitors (Sigma-Aldrich). The 10% SDS-PAGE gels separate total protein lysates and polyvinylidene fluoride (PVDF) membranes (Millipore, USA) transferred the protein lysates. The anti-IGF2BP3 antibody (1:1000, Proteintech, China), anti-β-Actin antibody (1:1000, Proteintech, China), and peroxidase (HRP)-conjugated secondary antibody (1:5000, Proteintech, China) incubated the membranes. After washing, signals were developed utilizing the chemiluminescence system (Bio-Rad, USA) and processed by Image Lab Software (NIH).