Fig 1: Global m6A levels in post-mortem brain tissuesGlobal m6A levels were measured by m6A RNA Methylation Assay Kit (Abcam: ab185912) according to manufacture instruction. Correlation analyses showed that global m6A levels were not significantly affected by PMI, pH, or RIN. However, there was a trend of decreasing global m6A levels with age.
Fig 2: NF90‐NF45 complex impairs m6A modification of pri‐mir‐7‐1 by METTL3/14 owing to preferential binding of NF90 to pri‐miRNAs in vitro. (A and B) Predicted structural models of pri‐mir‐7‐1/pre‐mir‐7‐1 (A) and pri‐mir‐200a/pre‐mir‐200a (B) by RNAfold (http://rna.tbi.univie.ac.at/cgi‐bin/RNAWebSuite/RNAfold.cgi) using the minimum free energy (MFE) model. Temperature conditions and salt concentration were 37 °C (default) and 1.021 m (default), respectively. The models were represented by FORNA (http://rna.tbi.univie.ac.at/forna). (C and E) In vitro m6A modification assay performed using pri‐mir‐7‐1 (C) and pri‐mir‐200a (E) probes and recombinant METTL3/14, NF90 and NF45 proteins. The levels of m6A modification on pri‐mir‐7‐1 or pri‐mir‐200a were detected by immunoblotting and standardized by methylene blue staining. The spot intensities were measured by densitometry and presented as a bar graph. Data are presented as a scatter plot and expressed as the mean ± SD [n = 4 (C) or 3 (E) per group]. (D) The level of m6A modification in pri‐mir‐7‐1 probes was analyzed using the m6A RNA methylation assay quantification kit in accordance with the manufacturer's protocol (ab185912; Abcam). The assay was performed using a pri‐mir‐7‐1 probe and recombinant METTL3/14, NF90 and NF45 proteins. Data are presented as a scatter plot and expressed as the mean ± SD (n = 5 per group). *P < 0.05, **P < 0.01 relative to control using a two‐tailed Welch's t test. ‘+’ and ‘++’ indicate 25 and 50 ng of recombinant protein, respectively.
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