8100 Nucleic Acids Research, 2011, Vol. 39, No. 18 Figure 6. Splicing of ESE depleted versions of HIPK3-T is not rescued by higher concentrations of nuclear Tra2b protein. (A) Two possible models of HIPK3-T splicing inclusion in response to Tra2b. (B) Graphs of dose responses of wild-type HIPK3-T and mutant HIPK3-T B1 and B3 to increasing concentrations of Tra2b (transfections of 300, 900 and 1500 ng of Tra2b expressing plasmid). (C) Levels of GFP-containing proteins were detected by a-GFP (left panel); Tra2b protein detected using a-Tra2b (middle panel); and a-tubulin (right panel) measured in parallel in transfected cells. The response of HIPK3-T to Tra2b is most consistent with an additive mode of splicing activation The results shown in Figures 2 and 5 indicate that only the combination of the four ESEs and the weak 50-splice site present in the wild-type HIPK3-T exon are able to induce a speciﬁc and efﬁcient on/off splicing switch in response to Tra2b expression. These results are consistent with two conceptually different models of HIPK3-T splicing regulation (Figure 6A). The ﬁrst ‘splicing threshold’ model predicts that concomitant occupancy of each of a minimum number of four ESE sites would be essential for HIPK3-T splicing activation. According this model, decreasing the ESE number would totally incapacitate the exon with regards to splicing activation and increasing Tra2b concentration within the cell should not be able to rescue splicing. The second ‘kinetic probability’ model is based on the probability of Tra2b occupancy of the HIPK3-T exon, through analogy with the model for Tra2 protein regula- tion of Drosophila Dsx splicing (17). In this model, multiple ESE sites would increase the probability of co-residency of Tra2b and U1 snRNP on the HIPK3-T exon, and ensure splicing activation on nascent RNA before the transcription of downstream competing exons