Anti fto

Combined with a protease inhibitor cocktail and phosphatase inhibitor cocktail, RIPA lysis buffer was employed to make cells lysed. Protein concentrations were measured with a BCA kit. Separated by SDS‐PAGE, the protein was transferred onto the PVDF membranes. 5% BSA was used for blocking up to 2 h, the membranes then were incubated with primary antibodies at 4°C overnight. After the incubation and the subsequent washing for 30 min, membranes were incubated with HRP Goat Anti‐Rabbit IgG or HRP Goat Anti‐Mouse IgG antibodies at room temperature. Primary antibodies were as follows: anti‐YTHDF3 (ab220161, 1:1000 dilution, Abcam), anti‐LOXL3 (sc‐377216, 1:100 dilution, Santa‐Cruz), anti‐β‐actin (#4970, 1:1000 dilution, Cell Signaling Technology), anti‐eIF3A (ab128996, 1:1000 dilution, Abcam), anti‐METTL3 (15073‐1‐AP, 1:1000 dilution, Proteintech), anti‐METTL14 (26158‐1‐AP, 1:1000 dilution, Proteintech), anti‐FTO (27226‐1‐AP, 1:1000 dilution, Proteintech), anti‐ALKBH5 (16837‐1‐AP, 1:1000 dilution, Proteintech), anti‐CHD7 (A17180, 1:1000 dilution, ABclonal), anti‐PDE3A (A17919, 1:1000 dilution, ABclonal).

Paraffin-embedded tissue sections were used for examination of hematoxylin-eosin (HE) staining. For IHC staining, tissue sections were repaired in 0.1 M sodium citrate buffer, then washed with PBS, and incubated in 3% hydrogen peroxide at room temperature for 25 min in darkness. Following blocking with 5% horse serum at 37 °C for 30 min, they then incubated with the following primary antibodies: anti-METTL3 (1:100, Proteintech, Wuhan, China), anti-FTO (1:100, Proteintech, Wuhan, China), anti-MTNR1B (1:100, Cell Signaling Technology, Danvers, MA, USA). Then, the slides were incubated with the secondary antibody. Chromogen detection was carried out with the DAB chromogen kit (Zhongshan Golden Bridge Biotechnology, Beijing, China). All images were taken using a microscope (Olympus, Tokyo, Japan).

Cells were collected and lysed in protein lysis buffer, and the concentration was measured using a BCA kit (Thermo Fisher Science, 23327). Protein samples (40 μg) were subjected to electrophoresis and transferred to nylon membranes, which were sealed with blocking buffer. Finally, the cells were incubated with the primary antibody at 4 °C for 6 h. The antibodies used were anti-FTO (cat: 27226-1-AP, Proteintech, China), anti-ALKBH5 (cat: 16837-1-AP, Proteintech, China), and anti-ARHGAP35 (cat: 26789-1-AP, Proteintech, China).

Enrichment of AlkBH5 or FTO in the IL-13 m⁶A region was further assessed using PureBindingRNA immunoprecipitation kit (Geneseed) according to the manufacturer’s instructions. In brief, the cells were lysed in RIP lysis buffer at 4 °C for 10 min, and the sample was divided into three parts (100 μl each), with two parts subjected to RIP with protein A/G sepharose beads conjugated with anti-AlkBH5 (5 μg, Proteintech) or anti-FTO (5 μg, Proteintech) antibody and normal rabbit IgG (Abcam) at 4 °C for 6 h. The RNA bound to the antibodies was purified and dissolved in RNASE-free water. RT-qPCR was employed to analyze the number of IL-13 transcripts in the immunoprecipitated RNA and total RNA from the whole cell lysates.