Ab117809

RIP experiments were performed using the Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) and the m⁶A antibody (Abcam, Cambridge, MA, USA) following the manufacturer’s protocol.
Briefly, cells (1×10⁷) were lysed with ice-cold RIP Lysis Buffer. Collect and store the cell lysate at -80 °C. Prepare the magnetic bead-antibody complex with 5 μg of the above-mentioned target antibody or control IgG and 50 μl of protein A/G magnetic beads, rotating at room temperature for 30 min. Take an equal volume of cell lysate and incubate the magnetic bead-antibody complex with rotation at 4 °C overnight so that the antibody can fully contact and bind to the protein. At the same time,10 μl cell lysate was extracted and used as input. Next day, RNA was extracted and purified with the prepared proteinase K buffer. Acquired RNA was used as a template to synthesize the corresponding cDNA.
Co-precipitated RNAs used for the first strand cDNA synthesis with the Reverse Transcription System (Roche) following the manufacturer’s protocol. Acquired cDNA was then analyzed by RT-qPCR. The information of IGF2BP1/2/3 antibody are ab184305, ab117809 and ab177477 (Abcam, Cambridge, MA, USA).

The interaction between IGF2BP2 and HPSE was analyzed by RIP assay following a previous report with some modifications [25 (link)]. RIP analysis was performed with a Magna RIP kit (Sigma-Aldrich). In brief, 1 × 10⁷ HaCaT cells were lysed in RIP lysis buffer, and incubated with the magnetic beads coated with IGF2BP2-specific antibodies (ab117809, 1:200 dilution, Abcam) for 6 h. IgG served as negative control. Coprecipitated RNAs were isolated, and HPSE enrichment level was detected.