Anti ythdc1 antibody

RIP assays were conducted using the Magna RIP Kit (Millipore, New Bedford, MA) according to the manufacturer’s protocols. Cells were prepared using RIP lysis buffer and the RNA-protein complexes were immunoprecipitated using anti-YTHDC1 antibody (PROTEINTECH, Rosemont, IL) and normal rabbit IgG. The co-precipitated RNAs were purified using phenol:chloroform:isoamyl alcohol and subjected to reverse transcription-PCR or real-time PCR analysis. A control amplification was carried out on the input RNA before immunoprecipitation.

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.