Anti u2af1 antibody

To create the MIG (MND Ires GFP)-U2AF1-Flag plasmid, U2AF1-Flag cDNA was amplified from the p3X-Flag-U2AF1 plasmid (obtained from the Kinji Ohno laboratory, Nagoya, Japan) and cloned into the MND-Ires-GFP (MIG) vector.^{24 (link)} The S34F, S34Y, S34F/Q157R, Q157R and Q157P mutations were generated by site-directed mutagenesis (Life Technologies) using the WT MIG-U2AF1-Flag construct as a template. 293T cells were co-transfected with each MIG-U2AF1-Flag expression plasmid described above and either the GH1 or FMR1 minigene reporter constructs described previously.^{1 (link), 25 (link)} GFP+ cells were sorted 48 hours later, followed by RNA extraction and RT-PCR as previously described.^{1 (link)} Amplicons were visualized by polyacrylamide gel electrophoresis and quantified by densitometry (ImageJ). Three independent experiments were performed for each assay and analyzed using the Student’s t-test. Lysates were made from transfected 293T cells and immunoblotting was performed using rabbit polyclonal anti-U2AF1 antibody (Abcam) to confirm over-expression.