Fig. 6. Dominant negative RhoA prevents hypoxia-induced Na,K-ATPase endocytosis. Permanently transfected A549 cells expressing either vector or dominant negative RhoA (RhoAN19) were grown in the presence of G418. (A) Actin stress fiber formation was assessed in DN-RhoA cells fixed and stained with rhodamin-palloidin and evaluated by using fluorescence microscopy. Representative photomicrographs of control cells (21% O₂), and hypoxia (1.5% O₂)-treated cells are shown. (B) Na,K-ATPase endocytosis was studied by biotin labeling of surface proteins followed by streptavidin pull-down and western blot analysis using a specific antibody after exposure to 1.5% O₂ for 60 minutes. Top: bars represent mean ± s.e.m. (n=3). ^**P<0.01. Bottom: a representative western blot. (C) Na,K-ATPase endocytosis was studied by biotin labeling of surface proteins followed by streptavidin pull-down and western blot analysis using a specific antibody after treatment with 100 µM H₂O₂ for 30 minutes. Top: bars represent mean ± s.e.m. (n=3). ^**P<0.01. Bottom: a representative western blot.