Fig. 4. Generation of KPT2-derived cell lines that express TRPV4. (A) In immunoblots, three empty vector control clones (KPT2-pBabe) did not express TRPV4, whereas three TRPV4-expressing clones (KPT2-TRPV4) highly expressed TRPV4 with two or three bands of 110 kDa. Actin bands at 42 kDa confirmed that there were similar amounts of total protein in the samples. (B) There was negligible TRPV4 expression in KPT2-pBabe cells. (C) In KPT2-TRPV4 cells, immunofluorescence showed uniform localization of TRPV4 at the cell membrane. (D) Orthogonal view of KPT2-TRPV4 shows that TRPV4 protein expression is uniform in the membrane, including the apical membrane. Scale bar in B also applies for C and D (20 µm). (E) TRPV4-dependent Ca²⁺ entry in response to hypotonic stress or the TRPV4 agonist 4-PDD was observed in KPT2-TRPV4 cells, but not in KPT2-pBabe cells. Fura-2 fluorescence was monitored as an index of intracellular Ca²⁺ concentration (see Materials and Methods), and the Fura-2 ratio (in arbitrary unit; U) is depicted as a function of time. Arrowhead denotes onset of hypotonic stress or addition of 4-PDD.