FIG 5.

m6A modification in HBx coding region affects HBx expression by interaction with YTHDF2. (A and B) Huh7 cells were transfected with pSI-X WT or A241T MT plasmid for 48 h. (A) Total RNA and cell lysates were extracted. m⁶A methylated RNAs were enriched by an anti-m6A antibody and normalized by input RNA levels using RT-qPCR. (B) The indicated proteins were assayed by immunoblotting. (C and D) Huh7 cells expressing pSI-X WT or pSI-X A241T MT were transfected with FLAG-YTHDFs expression plasmids for 48 h. The RNA-protein complexes were immunoprecipitated using an anti-FLAG antibody. (C) Immunoprecipitated HBx mRNA levels were normalized by input HBx mRNA using RT-qPCR. (D) The indicated proteins were analyzed by Western blotting. In panels A and C the error bars represent the SDs from three independent experiments. The P values were calculated via an unpaired Student's t test. *, P < 0.05; **, P < 0.01. IP, immunoprecipitation; n.s., nonsignificant; N.D., not detected.