n identified in CXCR4, the function of this nuclear targeting signal in the context of CXCR4 has not been examined. A distinct importin-dependent transport pathway has been implicated in the transport of C-C chemokine receptor type 2 . Favre et al. found that an engineered, HA-tagged CCR2 associated with a member of the importin family of nuclear transport receptors, Transportinb1, in a CCR2-null cell line. An interaction of CCR2 with TRN1 was required to detect CCR2 in nuclear fractions, suggesting that CCR2 transported to the nucleus via TRN1. TRN1 has been implicated in GPCR internalization and desensitization. Furthermore, TRN1 serves as a receptor for NLS-null proteins in NLS-containing cargo substrates, making it an essential protein for import through the nuclear pore complex. Taken together, these studies suggest that both the classical nuclear import machinery and TRN1 are candidates that play a role in CXCR4 nuclear translocation. 23300835 Nuclear CXCR4 protein expression has been observed in malignant 5-Carboxy-X-rhodamine web hepatocellular, colorectal, renal cell and nasopharyngeal carcinomas. These studies, however, were reported as clinical observations, and failed to investigate the mechanisms of CXCR4 localization or any biological function associated with the nuclear receptor. These data are consistent with reports that have demonstrated functional GPCRs associated with the nucleus, and further contribute to ongoing cancer therapeutic interventions against CXCR4. Importantly, a functional nuclear CXCR4 may contribute to PCa relapse despite current antagonists and monoclonal antibodies 25279926 against PM-bound CXCR4 and may not be designed to cross the PM, which would be required to antagonize active CXCR4 at the nucleus. Furthermore, identification of transport pathways required for nuclear localization of CXCR4 may reveal additional targets for therapeutic development to hinder prostate cancer metastasis and improve patient survival. Materials and Methods Cell Culture, Antibodies and Reagent Conditions PC3, DU145, 22RV1 human prostate cancer cell lines, RWPE1 human prostate cell line and 293T human embryonic kidney cell line were obtained from American 
Type Culture Collection. PC3, DU145, 22RV1 and 293T cells were maintained in complete RPMI media: RPMI 1640 containing 10% fetal bovine serum, 1% non-essential amino acids and 1% antibiotic-antimycotic at 37uC in 5% CO2. RWPE1 cells were maintained in keratinocyte serum-free medium containing 50 mg/ml gentamycin, 0.05 mg/ml bovine pituitary extract, and 5 ng/ml epidermal growth factor at 37uC in 5% CO2. All cells were maintained at 60% to 80% confluency. PC3 cells were originally isolated from a prostate vertebral metastasis, while DU145 cells were obtained from prostate brain metastasis. 22RV1 cells were from a human prostate carcinoma epithelial cell line derived from a xenograft that was serially propagated in mice, and RWPE1 cells were isolated from normal human prostate epithelium. Cell culture supplies and kanamycin sulfate were from MediaTech; SDF1a was from PeproTech. The following reagents and human antibodies were from Cell Signaling: 106 cell lysis buffer, mouse antirabbit IgG, anti-CD44, anti-GFP and anti-Gai. Anti-CXCR4 was from R&D Systems. Anti-Topoisomerase1, anti-Lamin A/C, Fusin -CXCR4, Fusin -CXCR4, anti-Fibronectin IgG2B, anti-GFP, Protein A/G Plus-Agarose beads, anti-Karyopherinb2, Karyopherinb2 siRNA and DAPI were from Santa Cruz Biotech. NE-PER Nuclear and Cytoplasmic Extraction Kit, Prote
