Ab186733

An EZ‐Magna RNA immunoprecipitation (RIP) Kit (Millipore, 17–701) was used according to the manufacturer's instructions. Briefly, 5 × 10⁶ BMSCs were lysed and magnetic beads conjugated with anti‐AGO2 (Abcam, ab186733, 1:50) or negative control IgG (Cell Signaling Technology, 2729S, 1:50) were added. The immunoprecipitated RNAs and the input RNAs were extracted and subjected to qPCR followed by electrophoresis to detect the expression of HHAS1.

An RNA immunoprecipitation (RIP) assay was conducted in breast cancer cells using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to manufacturer's instructions. Anti-Argonaute2 (AGO2) antibody (Abcam, USA, ab186733) or NC rabbit IgG (Cell Signaling Technology, USA, 7076P2) were used. Briefly, cells were collected and lysed by the RIP lysis buffer. Then the corresponding antibodies were added into the cleared lysates and incubated in the magnetic beads' suspension with rotating overnight at 4 °C. Precipitate was digested with proteinase K buffer, and then qRT-PCR was used to measure the levels of circMETTL3 transcripts in the AGO2 or IgG immunocomplexes.
The m6A RIP was performed as previously described with some modifications. Briefly, 3 μg of anti-m6A antibody (Abcam, USA, ab208577) was conjugated to protein A/G magnetic beads overnight at 4 °C. And then the antibody conjugated beads were incubated with the antibody in IP buffer with RNase inhibitor and protease inhibitor. The interacting RNAs were isolated and detected by qRT-PCR.

The experiment was performed as previously described.¹⁴ First, RIPA buffer was prepared, and cells were lysed for 30 min on ice. Then, the cell lysates were collected and centrifuged at 14,000 rpm for 30 min at 4°C. After that, the protein concentrations were measured, and equal amounts of proteins were mixed with loading buffer. The proteins were separated via electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, IPVH0010). Then the membranes were blocked with 5% non‐fat milk solution for 1 h. Thereafter, the membranes were incubated with primary antibodies followed by secondary antibodies. Finally, protein levels were detected using Chemiluminescent HRP Substrate (Millipore, WBKLS0500) and analyzed with ImageJ.
The following antibodies were used for western blotting: anti‐Osterix (Abcam, ab209484, 1:800); anti‐OCN (Abcam, ab93876, 1:800); anti‐AGO2 (Abcam, ab186733, 1:1500); anti‐RUNX2 (Cell Signaling Technology, 12556S, 1:1000); anti‐Collagen I (ColI; Abcam, ab260043, 1:1500); anti‐IRF2 (Abcam, ab124744, 1:2000); anti‐YY1 (Abcam, ab109228, 1:2000); anti‐GAPDH (CWBIO, CW0100M, 1:3000); and HRP‐conjugated secondary antibodies (Boster, 1:5000).