Fig. 6.
Hypoxia induces TRPV4 translocation into caveolae. Pulmonary artery smooth muscle cells were kept in room air (normoxia) or 1% O2 (hypoxia) for 15 min and then lysed. Lysates were subjected to sucrose density gradient ultracentrifugation, and seven fractions were collected from the resulting gradients. Each fraction was run on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and membranes were subsequently immunoblotted for TRPV4 and caveolin-1, as a marker for caveolae. (A) Representative Western blot images. Sucrose gradient fractions are shown in increasing density. The top row depicts TRPV4 immunoblots in normoxia and hypoxia, as indicated. Bottom panel shows caveolin-1 (cav-1) as a marker of caveolae. Note the leftward movement of TRPV4 during hypoxia into cav-1-containing fractions. Black bar indicates the fractions considered to be caveolae for subsequent analysis. (B) Densitometric analysis was performed, and the fraction of TRPV4 signal in the caveolin-1-containing fractions was expressed relative to total TRPV4 signal in all fractions. Data shown are ± SEM for n = 2 experiments. *P = 0.01.