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Figure 7


Fig. 7. ROCK activation is necessary in hypoxia-induced Na,K-ATPase endocytosis. (A) A549 cells were exposed to hypoxia in the presence or absence of 10 µM Y-27632 and cell lysates were obtained. Equal amount of proteins were separated by SDS-PAGE and immunoblotted with either phospho-MYPT (Thr696) antibody or pan-MYPT antibody. A representative western blot is shown (n=3). (B) A549 cells were pre-incubated in the presence of vehicle (V) or 10 µM Y-27632 and then exposed to hypoxia for 60 minutes. 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. Top: bars represent mean ± s.e.m. (n=3), **P<0.01. Bottom: a representative western blot. (C) COS-7 cells were transiently transfected with vector, DN-ROCK, and Na,K-ATPase endocytosis was studied by biotin labeling of surface proteins. Top: bars represent mean ± s.e.m. (n=3), **P<0.01. Bottom: a representative western blot for {alpha}1-Na,K-ATPase endocytosis and Myc-ROCK expression levels.





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