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Expression of UT‐B urea transporters in the RT4 urothelial cell line
Author(s) -
Stewart Gavin,
Farrell Alan
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.857.30
Subject(s) - urea , gene isoform , western blot , blot , microbiology and biotechnology , cell culture , biology , apoptosis , urothelial cell , transporter , chemistry , bladder cancer , gene , biochemistry , genetics , cancer
Facilitative UT‐B urea transporters have been localized to the urothelial cell layer lining the bladder in various mammals, including humans (Walpole et al., 2014). Previously, two reports have associated UT‐B allelic variation with bladder cancer risk (Garcia‐Closas et al., 2011; Rafnar et al., 2011) and UT‐B knockout mice are known to suffer DNA damage and apoptosis in the bladder (Dong et al., 2013). The aim of this current study was to investigate whether the RT4 cell line was an appropriate cell model for investigating human urothelial UT‐B urea transporters. End‐point PCR experiments were initially performed to investigate the expression of various urea transporters previously found in the human bladder. As expected, the RT4 cells were shown to express UT‐B1, UT‐B2 and AQP3. Next, western blotting experiments were performed to investigate for protein abundance. While no protein was detected for AQP3, strong protein signals were indeed detected for UT‐B urea transporters. A strong 40–70 kDa, smeared UT‐B signal was detected in membrane‐enriched protein samples, but not in cytosolic‐enriched samples. Using a one hour, 37°C pre‐incubation with PNGaseF enzyme, this UT‐B smear was shown to deglycosylate to a strong 28 kDa band and a weak 40 kDa band. These data show that UT‐B urea transporters are highly expressed in RT4 cells, with both PCR and western blot analysis indicating multiple UT‐B isoforms are present. These findings suggest that RT4 cells may be an appropriate model for investigating human urothelial UT‐B urea transporters. Support or Funding Information This work was supported by The Jack Pickard Research Foundation and University College Dublin.UT‐B urea transporter protein in RT4 cells.