Journal: Nature Communications
Article Title: BEX1 is an RNA-dependent mediator of cardiomyopathy
Figure Lengend Snippet: BEX1 directly controls A + U-rich element containing mRNAs. a Comparison of RNAseq and RIP-seq list showing 91 overlapping mRNAs due to BEX1 overexpression or BEX1-dependent pull-down, respectively, 17.58% of which contains AU-rich elements (ARE). b RNA EMSA showing an ARE-ribonucleoprotein cardiac complex shift from heart protein extract at 4 and 8 μg, which is shifted even higher by addition or recombinant BEX1-GST (blue arrows). GST was used as negative control. c qPCR for β-globin mRNA at 0, 45, 90, 135 and 180 min post-tetracycline addition in stable cell lines (control, GM-CSF ARE or c-fos ARE) transfected with BEX1 or GFP control plasmid. A synthetic poly A (SPA) is present in all 3 constructs. Results reflect 3 separate experiments. d Level of β-globin mRNA following RNA immunoprecipitation using Flag antibodies from Flag-BEX1 or GFP expressing stable cell lines (control, GM-CSF ARE and c-fos ARE) by qPCR. The results are from 3 separate experiments. e qPCR analysis for TNFα and CCL5 mRNA immunoprecipitated with BEX1 antibody from BEX1 transgenic hearts. Number of experiments conducted is shown in the graph for each condition. * P
Article Snippet: For RIP-sequencing, Magna RIP kit (Millipore) was used to extract RNA, and anti-Flag M2 magnetic beads (M8823, Sigma) were used to immunoprecipitate ribonucleoprotein complexes from neonatal rat cardiomyocytes overexpressing Flag-tagged BEX1 or β-galactosidase control.
Techniques: Over Expression, Recombinant, Negative Control, Real-time Polymerase Chain Reaction, Stable Transfection, Transfection, Plasmid Preparation, Construct, Immunoprecipitation, Expressing, Transgenic Assay