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Figure 2


Fig. 2. New kidney cell line generation and TRPV4 expression. By spontaneous immortalization, three cell lines were generated from primary murine kidney cultures. (A,B) Bright-field images show KPT2 cells with a fibroblastic morphology (A), and KDT3 cells with an epithelial (cobblestone-like) appearance and smooth edges to cell clusters (B). (C,D) TRPV4 expression levels were determined by RT-PCR (C) and immunoblotting (D). KDT3 cells showed high levels of TRPV4 whereas KPT1 and KPT2 showed weak TRPV4 expression or possibly non-specific bands. Lower levels of β-actin following RT-PCR (C) and of actin following immunoblotting (D) suggest that KDT3 cells express β-actin and possibly other actin isoforms at lower levels compared with KPT1 and KPT2. (E) Expression of TRPV4 was negligible in KPT2 cells. (F) Immunofluorescence showed TRPV4 localization at the basolateral membrane (arrowheads) of KDT3 cells. (G) Orthogonal view of KDT3 cells shows TRPV4 expression in the basolateral membrane (arrowheads). Scale bars, 20 µm.





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