Anti-ac4C dot blot assays were performed as previously described (20 (link)). Briefly, 5 μg RNA was denatured for 5 min at 75°C, placed on ice for 1 min, loaded onto Hybond-N+ membranes, and subjected to ultraviolet crosslinking. ac4C detection was carried out with anti-ac4C antibodies (Abcam, Cambridge, UK) using standard protocols. Signals were detected using a ChemiDoc MP imaging system (Bio-Rad Laboratories, Berkeley, CA, USA).
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