Fig 1: BFA reduces expression of SRSF9 and Tra2-β, and enhances SMN exon 7 ESE-binding activity of hnRNP M and PSF. (A) SMA fibroblasts were treated with BFA (0.1 and 1 µM) for 24 h, and total cell lysates were prepared and subjected to Western blotting with anti-SRSF1, -SRSF9, -Tra2-β and -hnRNP A1 antibodies. (B) The amounts of SRSF1, SRSF9, Tra2-β and hnRNP A1 proteins in panel a were quantified using ImageJ software. The relative amount of each protein was calculated by normalizing to the value from DMSO-treated SMA cells and presented as a percentage. The mean values and standard deviation were determined from two independent experiments. (C) The SMN2 exon 7 splicing effected by siRNA-mediated knockdown of splicing factors was analyzed by RT-PCR. siRNAs targeting GFP mRNA and scrambled siRNA were used as controls. GAPDH mRNA was also analyzed as a control. (D) The PCR products of FL-SMN and SMNΔ7 mRNAs in panel c were quantified using the ImageJ software. The relative amount of each PCR product was determined by normalizing to the value from GFP siRNA-transfected SMA cells, and are presented as a percentage. (E) 293T cells were treated with DMSO or BFA (0.1 μM) for 24 h, and the cell lysates were prepared and subjected to an RNA affinity assay. Cell lysates were incubated with biotinylated Exon 7-ESE RNAs, and ESE-bound proteins were precipitated by biotin-streptavidin bead interaction. Precipitated proteins were analyzed by Western blotting with anti-PSF, -hnRNP M, -Tra2-β, -hnRNP G and -SRSF9 antibodies. Histone H3 was also analyzed as a negative control.