FIG. 6.

Overexpression of PTB in DT3 cells leads to a reduction in exon IIIb inclusion. (A) Transient transfection of a HisG-tagged CMV-driven full-length PTB1 expression plasmid results in high levels of PTB expression in DT3 cells. Western analysis was performed following transfection with empty vector or 0.5, 1.0, or 2.0 μg of PTB. The gels were probed for nuclear protein CA150, as a loading control, HisG, or PTB as indicated. The formation of the PTB triplet seen at higher levels of overexpression could represent a degradation product, an internal translation start site, or an effect on endogenous PTB alternative splicing. (B) Transient transfection of progressively increasing amounts of HisG-PTB (in nanograms) results in progressively decreased amounts of IIIb inclusion. (C) Comparison of PTB-mediated repression using 500 ng of HisG-PTB in the presence (+ISS) or absence (ΔISS) of ISS. The results are normalized to represent the percentage of IIIb skipping relative to that in cells transfected with empty vector. (D) Quantified averaged experiment of a set of triplicate transient cotransfections of the three PTB splice variants with a minigene that contains ISS. Exon IIIb inclusion has been normalized by making the vector-alone level of inclusion equal to 100 and then representing all other values as a percentage of that amount. Averaged values of triplicate experiments are shown, and the standard deviation was less than 10% for all data points. These splice variants were HA tagged and lacked the first 56 amino acids; however, full-length splice variants yielded identical curves (not shown). (E) Transfection of the same expression plasmid expressing HisG-tagged LacZ reveals high levels of expression of an unrelated protein by immunoblotting. (F) Cotransfection of LacZ does not alter the level of exon IIIb inclusion when cotransfected with pI-11-IIIb:+ISS+ISAR.