There is thus a huge need for innovative and new treatment strategies

ese nuclear body components tagged with YFP were used to co-transfect avian and human cells with pGag.L219A-CFP. We expected human SC35 and SF2 to form characteristic splicing speckles in QT6 cells due to the high level of conservation between human and chicken orthologs. However, SC35 and SF2 appeared more diffuse in QT6 cells even when a low amount of plasmid DNA was used for transfection, although there were areas of consolidation where the protein was concentrated. Of interest, both SC35 and SF2 co-localized with Gag.L219A foci to a high degree. Quantitative Mander’s analysis performed in 8 cells revealed that a mean of 69.8 4.7% of Gag.L219A co-localized with SC35 and 61.5 5.2% of Gag.L219A with SF2. To determine whether co-localization was present in 3-dimensions, z-stacks were obtained and reconstructions were performed using Imaris imaging analysis software. Rendering of the Gag.L219A and SC35 or SF2 signals revealed that Gag.L219A/SC35 and Gag.L219A/SF2 co-localized in the x, y, and z dimensions and appear to be in close proximity, at least based on the limits of resolution of the microscopic images obtained. For Gag.L219A/SC35 and Gag.L219A/SF2, the Mander’s co-localization values were statistically significantly higher than the quantitative co-localization measured between Gag.L219A and proteins that reside in paraspeckles or PML bodies . Together, these data suggest that Gag.L219A protein Peretinoin accumulated at subnuclear locations enriched in splicing speckle components SC35 and SF2. Next, because we observed co-localization between Gag.L219A and human SC35 and SF2 expressed in avian cells, we wanted to determine whether similar co-localization patterns would be observed in human cells, in which the localization of splicing factors has been more extensively studied. When expressed alone, SC35 adopted its characteristic speckled appearance; however, co-expression with Gag.L219A resulted in a more diffuse pattern, and there was a high degree of co-localization between the proteins. SF2 accumulated in speckles when expressed alone, and SF2 also showed a high degree of co-localization with Gag.L219A. Gag co-localization was significantly higher with SC35 compared to SF2 and both SC35 and SF2 were more strongly associated with Gag.L219A compared to p54nrb, PSF or PSP1. Analysis of cells co-expressing Gag.L219A and PML or SUMO1 could not be performed due to cell toxicity. To determine whether Gag.L219A foci co-localized with endogenous nuclear splicing speckles, we performed immunofluorescence using an PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19815280 antibody that recognizes the phosphorylated RS domains of SF2 and SC35. Gag.L219A accumulated in nuclear foci in transfected HeLa cells that appeared similar to those observed in QT6 cells. In cells expressing Gag.L219A, overlapping signals were observed at the intersection of the phosphorylated SR domain proteins and Gag.L219A foci, which appeared to be juxtaposed, suggesting that Gag foci form near accumulations of splicing speckle components. Of note, in HeLa cells expressing Gag.L219A, the amount of endogenous SC35/SF2 staining was dramatically reduced, as indicated by the statistically significant decrease in the mean signal intensity of the anti-phospho RS antibody channel compared to cells in which there was no Gag expression. This result suggests that expression of Gag.L219A interferes with staining of endogenous phospho RS domains of splicing factors, although the mechanism remains unclear. Co-expression of Clk1 Enhances Co-locali