Figure 3.

PfSR1 is an AS factor with similar activity to SRSF1. (A) Schematic illustration of the experimental procedure designed to demonstrate AS activity of PfSR1. HEK 293 T cells were cotransfected with plasmids that carried a mini-gene as well as an expression vector (pcDNA3⁻). Forty-eight hours post-transfection the cells were harvested and their RNA and protein content were analyzed. (B) Western blot analysis using anti-T7 antibody showing the expression of the tagged SRSF1 (Lane 2) and PfSR1 (Lane 3). An empty pcDNA3⁻ vector was used as a negative control (Lane 1). (C) RT–PCR amplification of total RNA extracted from HEK 293 T cells that were cotransfected with the E1A mini-gene obtained differential splicing patterns mediated by PfSR1 and SRSF1 compared with the control. Schematic of the mini-gene and the differentially spliced isoforms is shown on the left. GAPDH was used as loading control and is presented in the lower panel. (D) Quantification of the changes in the proportion of each spliced isoform of E1A obtained after transfection with PfSR1 and SRSF1 compared with the control. The density of each band is presented as a proportion of the total signal obtained. The averages and standard errors of three different experiments are presented. (E) RT–PCR amplification of total RNA extracted from 293 T cells that were cotransfected with the β-globin mini-gene obtained differential splicing patterns mediated by PfSR1 and SRSF1 compared with the control. Schematic of the mini-gene and the differentially spliced isoforms is shown on the left. GAPDH was used as loading control and is presented in the lower panel. (F) Quantification of the changes in the proportion of each spliced isoform of β-globin obtained after transfection with PfSR1 and SRSF1 compared with the control. The density of each band is presented as a proportion of the total signal obtained. The averages and standard errors of two different experiments are presented.