Fig 1: Western blotting experiments showing effects of NaCl‐induced increases in external osmolality on protein abundance. (a) Increases in both 28 and 35–70 kDa UT‐B signals were observed with NaCl treatment, whereas there was no change in NaKATP signals. (b) Significant increases were also observed in 45 and 100 kDa AQP3 protein, with no effect observed for 37 kDa GAPDH. (c) Summary graph comparing the densitometry values in control (~300 mOsm) to +200 mOsm NaCl (~500 mOsm) treatments in all experiments for UT‐B (n = 7) and AQP3 (n = 7). (d) Bar graphs illustrating mean densitometry values for GAPDH, NaKATP, UT‐B and AQP3 after +100 and +200 mOsm NaCl‐induced treatments. Key: * = p < .05, ANOVA; ** = p < .01, ANOVA
Fig 2: Western blotting experiments showing effects of mannitol‐induced increases in external osmolality on protein abundance. (a) No changes in 28 or 35–70 kDa UT‐B signals were observed with mannitol treatment. In addition, there was no change in 100 and 150 kDa NaKATP protein signals. (b) Significant increases in 45 and 100 kDa AQP3 protein signals were detected with both +100 and +200 mOsm mannitol, while no such effect was observed for 37 kDa GAPDH. (c) Summary graph comparing the densitometry values in control (~300 mOsm) to + 200 mOsm mannitol (~500 mOsm) treatments in all experiments for both UT‐B (n = 4) and AQP3 (n = 5). (d) Bar graphs illustrating mean densitometry values for GAPDH, NaKATP, UT‐B and AQP3 after +100 and +200 mOsm mannitol‐induced treatments. Key: * = p < .05, ANOVA
Fig 3: End‐point RT‐PCR experiments showing UT‐B1 is the main UT‐B transcript present in the RT4 human urothelial cell line. (A) End‐point RT‐PCR experiments using a variety of primers showed that RT4 cells strongly expressed both UT‐B (F4/R5) and AQP3, but not AQP7 or AQP9. In contrast, human bladder was confirmed to highly express UT‐B, AQP3, AQP7, and AQP9. (B) Further experiments revealed that UT‐B1 was the predominant transcript in RT4 cells. Isoform‐specific primers (F1/R5) mainly detected UT‐B1, rather than UT‐B2. However, some UT‐B2 expression did occur, as confirmed through a set of UT‐B2‐specific primers (F3/R5). Key: + = reverse transcriptase present; - = reverse transcriptase absent
Fig 4: End‐point RT‐PCR experiments showing urea‐induced changes in external osmolality increase UT‐B RNA expression. Urea‐induced increases in osmolality appeared to increase both UT‐B1 and UT‐B2 expression, with the greatest increase observed in UT‐B1 with +200 mM urea. In contrast, no such increase was seen for AQP3, NaKATP, or actin. [Data shown are representative of two experiments performed and is therefore not quantitative.]
Fig 5: Representative colocalization analysis using the Leica LAS-AF image system. (A) Pixels distribution scatter plot: colocalized yellow pixels between the two diagonals, red (AQP8) pixels in ordinate and green (HPV) pixels in abscissa. (B) Leica LAS-AF software generates an image of colocalized pixels, highlighted in white, superimposed on an RGB-merge of two channels. (C) Overall colocalization values obtained from the representative colocalization analysis using the Leica LAS-AF image system. (D) Statistical analysis of Pearson’s coefficient R values was obtained from at least 6 different double immunofluorescence experiments with anti-HPV antibody and anti-AQP3, AQP7, or AQP8 antibodies. Negative controls (Neg) were also analyzed. a, p < 0.0001 vs. AQP3, AQP7 and Neg (ANOVA, followed by Newman–Keuls’s Q test).
Supplier Page from MilliporeSigma for Anti-AQP3 antibody produced in rabbit