Fig. 3. Hypoxia-induced activation of RhoA in A549 cells. (A) Serum-starved A459 cells were exposed to hypoxia for 15, 30 or 60 minutes, 1% Triton X-100-soluble membrane fractions were obtained and RhoA translocation was evaluated by western blot. Top: a representative western blot for RhoA translocation and total ₁-Na,K-ATPase as loading control; Bottom: points on the curve represent mean ± s.e.m. (n=3). ^*P<0.05; ^**P<0.01. (B) A549 cells were exposed to hypoxia for 5, 15, 30 or 60 minutes and cell lysates were subjected to a pull-down assay with a GST-tagged fusion protein rhotekin Rho binding (RBD) domain. A representative western blot of RhoA bound to GTP (top) and total RhoA (bottom) is shown (n=3). (C) A549 cells were plated onto glass coverslips and exposed to 21 or 1.5% for 15 or 60 minutes. After incubation cells were washed, fixed, stained with rhodamine-phalloidin and evaluated using fluorescence microscopy. Representative images are shown.