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. Author manuscript; available in PMC: 2020 Jun 16.
Published in final edited form as: Cell Rep. 2019 Sep 24;28(13):3435–3449.e5. doi: 10.1016/j.celrep.2019.08.060

Figure 4. Exons Upstream of FUBP1-Regulated Splice Sites Exhibit Diminished m6A Levels in FUBP1-Null Cells.

Figure 4.

(A) Western blot validation of significant proteins from IP/MS experiment.

(B and C) Dot blot measuring global m6A levels in mRNA of indicated cell lines (B), quantified in (C).

(D) RNA-IP with m6A-modified or non-modified RNA bait followed by immunoblotting for a bona fide m6A reader, YTHDF2, and FUBP1.

(E) Distribution of m6A-seq peaks across the CASP8, BRCA1, and MAGI3 loci, based on analysis of previously published m6A-seq data in HepG2 cells. The locations of the putative m6A sites are indicated within exons directly upstream of splice sites yielding AS transcripts found in FUBP1-null cells.

(F) Relative m6A levels at m6A consensus sites of CASP8, BRCA1, and MAGI3 in exons upstream of splice sites that yield alternative variants, determined by m6A RIP-qPCR in NTC and FUBP1-null MCF10F cells.

(G) CASP8, BRCA1, and MAGI3 mRNA levels relative to GAPDH determined by quantitative real-time PCR in NTC and FUBP1-null MCF10F cells (n = 2) using primers flanking the regions distal from splice sites, not surrounding m6A consensus sites. Data are presented as means ± SEM, n = 3 biological replicates per cell line. *p < 0.05 (two-tailed Student’s t tests), unless otherwise stated.