Ab207997

ChIP assay experimentation was conducted with the help of EZ-ChIP kits (Merck Millipore Corp., Billerica, MA, USA) [29 (link)]. Briefly, H9c2 cells were cross-linked with 1% formaldehyde at room temperature for 10 min and then quenched in 125 nM glycine for 5 min. Subsequently, DNA fragments with an average length of 0.5–2 kb were obtained by ultrasonification of the cross-linked chromatin. Next, the cross-linked protein-DNA complexes were precipitated using an anti-H3K9me2 antibody (dilution ratio of 1:100, ab1220, Abcam) and immunoglobulin G (dilution ratio of 1:500, ab207997, Abcam). qRT-PCR was carried out to analyze the precipitated DNA. PCR primer for YAP1 gene promoter region was as follows: Forward: 5’-CCCTGCCGGACCGGCGCG-3’ and Reverse: 3’-TGCAGATATTTTGTCCAA-5’.

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.

To perform immunofluorescence staining on the colon tissue sections, the frozen sections embedded in OCT were fixed in 8% neutral buffered formalin for 30 min. After fixation, the sections were washed three times with distilled water (ddH2O) to remove residual formalin. For the staining process, primary antibodies specific to the target proteins of interest, such as anti-mouse Muc2 (ab76774, Abcam), CRAMP (sc-66843, Santa Cruz), or β-Defensin 2 (ab203077, Abcam), Ki67 (ab16667, Abcam), IL-1β (AF-401-NA, R&D) antibodies and anti E. coli antibody (ab25823, Abcam), were applied to the sections. Following the primary antibody incubation, secondary antibodies conjugated with biotin (ab6720, ab208000, ab207997, ab207996, Abcam and BAF109, R&D), were applied to the sections followed with streptavidin-FITC (405202, BioLegend) or PE (405204, BioLegend), allowing for the visualization of the target proteins. The 6-diamidino-2-phenylindole (DAPI) was used to stain the cell nuclei. The fluorescence intensity of all immunofluorescence staining was measured by ImageJ.