Investigation of the demethylation impact of Dem6A-Vec expressing a catalytically inactive ALKBH5 (dALKBH5) after site-directed mutagenesis leading to the H204A mutant enzyme. (A) Sanger sequencing results validating the successful generation of Dem6A-Vec expressing dALKBH5. (B) SELECT-qPCR results for m⁶A:2886 of EEF2 and m⁶A:418 of RRAGA on WT HeLa cells and cells transfected with Dem6A-Vec expressing dALKBH5. The m⁶A stoichiometry on both conditions was calculated based on the 2^−ΔΔCt methodology, using a nonmethylated A site of HPRT1 gene as control.