Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides
Fig 7
Detection of m6A-demethylation by immuno-northern blotting.
(A) Demethylation of m6A to A in RNA by FTO and ALKBH5. (B) m6A-containig 15 mer-ssRNA was treated with or without FTO and analyzed by INB with anti-m6A antibody and SYBR. The bottom graph shows a densitometric quantification of the band intensity. Data were expressed relative to the mean value of the FTO (-) samples, which was taken as 1.0 AU, N = 3. (C) m6A-containig 15 mer-ssRNA was treated with or without ALKBH5 and analyzed by INB and SYBR. A representative image is shown. (D) LC-MS/MS analysis of FTO-mediated demethylation. After the FTO treatment, m6A-ssRNA was digested and subsequently analyzed by LC-MS/MS. Chromatograms of the m6A peak of digested nucleosides are shown. The right graph shows a quantification of the relative area ratio of m6A/A. Data were expressed relative to the mean value of the FTO (-) samples, which was taken as 1.0. (E) Knockdown of FTO and ALKBH5 in HeLa cells. The knockdown efficiency was measured by quantitative PCR. Relative abundance of each transcript was normalized by GAPDH. Data were expressed relative to the mean value of the control, which was taken as 1.0, N = 3. (F) Evaluation of the m6A modification in RNAs in FTO- and ALKBH5-knockdown HeLa cells. The mRNA (100 ng) and total RNA (1.0 μg) were analyzed by INB with anti-m6A antibody using acrylamide gel separation. Arrowheads denote the anti-m6A positive signals. SYBR staining is shown for the loading control. Values are shown as the mean ± SD. *P < 0.05.