Dot Blot Assay for ac4C Detection

ac4C detection by dot blot assay were conducted as described [8 (link)]. Total RNAs were extracted from A549 or H1299 stable cell lines and in vitro ac4C formation assays by TRIZOL reagent, the RNAs were denatured at 95 °C for 5 min and then immediately put on ice for 2 min. Equal amounts of serial-diluted RNAs were added into an N+ nylon membrane (GE Healthcare) and then crosslinked by 480 mJ/cm² at 254 nm UV light. The membrane was blocked with 5% milk in 1 × PBST for 30 min at room temperature and then inducted with anti‐ac4C antibody at 4 °C overnight. After washed three times by PBST the membrane was incubated with anti-rabbit IgG HRP conjugated secondary antibody for 1 h at room temperature, the membranes were finally imaged and analyzed. Methylene blue staining was used to verified that equal amount RNAs was spotted on the membrane.