expression of CXCR4, we evaluated the intracellular distribution of wildtype GFP-tagged CXCR4, two mutated fusion proteins in which arginine 146 and 19276073 148 were separately mutated to an alanine, as well as a fusion protein where the NLS was deleted. Plasmids encoding GFP-CXCR4 were transfected into PC3 cells and examined by ICC microscopy. The localization pattern of GFP-CXCR4 at the plasma membrane and in the cytoplasm was consistent with endogeneous CXCR4. Previous studies have reported an expression pattern for GFPCXCR4 similar to our observation in other cancer cell lines. Gynostemma Extract web Wild-type GFP-tagged CXCR4 was localized predominantly at the PM, with some localization at the nucleus in untreated cells. However, an increase in punctate staining was observed at the nucleus/nuclear membrane upon treatment with SDF1a. Interestingly, both CXCR4R146A and CXCR4R148A were detectable at the nucleus, suggesting that neither R146A nor R148A mutations in the NLS were sufficient to inhibit CXCR4 localization to the nucleus. To further examine the requirement of this NLS to localize CXCR4 to the nucleus, we deleted the NLS, `146RPRK149′, within pEGFPN1-CXCR4. We detected CXCR4DNLS at the PM and diffusely throughout the cytosol, similar to wild-type Nuclear CXCR4 in Metastatic Prostate Cancer Cells 8 Nuclear CXCR4 in Metastatic Prostate Cancer Cells GFP-CXCR4, but we did not detect CXCR4DNLS at the nucleus. To further confirm that CXCR4DNLS was excluded from the nucleus, PC3 cells were transiently transfected with wildtype GFP-CXCR4 or CXCR4DNLS then fractionated into nuclear and non-nuclear samples for analysis by western blot analysis. Consistent with ICC observations, we found that wild type GFP-CXCR4 and CXCR4DNLS were both detectable in non-nuclear fractions, while only GFP-CXCR4 was detected in nuclear fractions. Collectively, these data suggest that the `RPRK’ motif may be involved in localization of CXCR4 to the nucleus in prostate cancer. CXCR4 Demonstrated an Interaction with Transportinb1 We identified a putative NLS motif that could be critical for CXCR4 nuclear localization; however, the motif `RPRK’ is not a typical classical NLS. In fact, such sequences can also mediate direct binding to other transport receptors. Among the different molecules that are involved in the transport of various cargos to the nucleus, members of the karyopherin beta family contribute directly or indirectly to the nuclear shuttling of molecules. Transportinb1, 
also known as Karyopherinb2, is a transport molecule of the importin-b family that has been linked to desensitization and nuclear-cytoplasmic shuttling of receptors. To test whether TRN1 was involved in CXCR4 transport to the nucleus, we first established that PC3 cells expressed TRN1 by western blot analysis; 293T cells served as a positive control for TRN1 expression. Next, we tested for an interaction between CXCR4 and TRN1. We immunoprecipitated CXCR4 from whole cell lysates and tested for co-purification of TRN1 by western 22440900 blot analysis. to assess whether nuclear-associated CXCR4 was active. Whole cells were stimulated with SDF1a then harvested to isolate intact nuclei. Nuclei were lysed, then immunoprecipitated with anti-CXCR4, prior to immunobloting for associated Gai. In untreated cells, we observed a basal level in Gai expression, which decreased upon treatment with SDF1a, suggesting that nuclearassociated CXCR4 is functional and can respond to SDF1a. We further tested the functionality of nuclear-asso
