Ab286164

Proteins were first extracted using the radioimmunoprecipitation assay lysis solution with phenylmethylsulfonyl fluoride and protease inhibitor cocktail (APEXBIO, USA) and then measured using a bicinchoninic acid kit (Beyotime, China). SDS-polyacrylamide gel electrophoresis was used to separate the protein lysates, which were then transferred to polyvinylidene fluoride membranes. After blocking the membranes with 5% skim milk, they were incubated with specific primary antibodies overnight at 4 °C, followed by a 1-h incubation with secondary antibodies at room temperature. On the Mini-REPORT Tetra Electrophoresis System, proteins isolated on the membranes were visualized using an ECL chromogenic kit (Beyotime, China). The antibodies used in this study were as follows: FTO (Abcam; ab126605, 1:1,000), m6A (Abcam; ab286164, 1:1,000), METTL3 (Abcam; ab69325, 1:1,000), vimentin (CST; 5741T, 1:1,000), CDH1 (CST; 3195S, 1:1,000), Snail (Wanlei; WL01863, 1:1,000), IGF2BP2 (Proteintech; 11601-1-AP, 1:1,000), IGF2BP3 (Proteintech; 14642-1-AP, 1:1,000), DDIT4 (Proteintech; 10638-1-AP, 1:1,000), AKT (CST; 4685S, 1:1,000), TSC2 (Proteintech; 24601-1-AP, 1:1,000), p-AKT (CST; 13038S, 1:1,000), p-TSC-2 (Proteintech; 29000-1-AP, 1:1,000), AR (Proteintech; 22089-1-AP, 1:1,000), and GAPDH (Proteintech; HRP-60004, 1:1,000).

M⁶A enrichment was determined using the Magna MeRIP m6A Kit (Millipore, Billerica, MA, USA) following the manufacturer’s instructions. In brief, 18 μL of total RNA at a concentration of 1 μg/μL was mixed with 2 μL of Fragmentation Buffer 10× and heated at 94 °C for 5 min. After all the RNA was fragmented, size distribution was checked on 1.5% agarose gel. Magna ChIP Protein A/G Magnetic Beads (Millipore) were prewashed and incubated with anti-m⁶A antibody (Abcam, ab286164) or rabbit immunoglobulin G (IgG) for 30 min at room temperature. The immunoprecipitation mixture was prepared by incubating the beads above with fragmented RNA for 2 h at room temperature. The mixtures were placed on a magnetic separator, and methylated mRNAs were eluted using Elution Buffer, 2 μg of the RNA was served as the input, and the relative m⁶A-circUHRF2 enrichment normalized to input was determined by qRT-PCR.

The direct binding relationship between METTL3 and TRPC6 was assessed using a commercial RIP kit (Millipore). In detail, rRAECs (5 × 10⁷) were collected after different treatments and lysed utilizing RIP lysis buffer. The magnetic beads were preincubated with the specific antibodies targeting METTL3 (ab195352, Abcam), m6A (ab286164, Abcam), and mouse IgG (ab207997, Abcam) for overnight. Then, the antibody-bead complex was incubated with the prepared cell lysates at 4°C for 24 h. After proteinase K treatment, precipitated RNA was extracted and subjected to the RT-qPCR assay.

RIP assay was carried out using Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s protocol. Briefly, after centrifugation, the supernatant of cell lysate was inoculated with primary antibodies-conjugated beads overnight at 4 °C. Next, the binding RNAs were purified and analyzed by RT-QPCR. The primary antibodies including anti-N6-methyladenosine (m6A) (ab286164, Abcam), anti-METTL3 (ab195352, Abcam), Anti-YTHDC1 (ab220159, Abcam), Anti-FAM120A (ab156695, Abcam) and IgG (Abcam) were used.