Fig. 6.

Hyperoxia induces Cav-1 phosphorylation via ROS. Beas-2B human bronchial epithelial cells and primary mouse lung epithelial cells were used in these experiments. (A) Hyperoxia and H₂O₂ induce Cav-1 phosphorylation. Primary lung epithelial cells derived from C57BL/6 mice were incubated with 90 µM H₂O₂ for 20 min, exposed to hyperoxia (4 h), or left untreated. Cell lysates were collected and analyzed by Western blotting and were blotted with anti-PY14Cav-1. (B) NAC blocks hyperoxia-induced Cav-1 Y14 phosphorylation. Primary lung epithelial cells were pretreated with NAC (30 nM), which was followed by exposure to hyperoxia (4 h). Western blot analysis was used to determine Y14 phosphorylation. Similar results were found in Beas-2B cells. (C) Beas-2B cells were transfected with GPX2 siRNA or control siRNA (left) or with catalase overexpression clones or control vectors (right). After 24 h, cells were exposed to hyperoxia (4 h). Coimmunoprecipitation of Cav-1 and BID was determined. Cell lysates were incubated with anti-BID antibody overnight at 4 °C and then coupled with protein A/G agarose beads. The Cav-1 protein, which coimmunoprecipitated with BID, was detected by Western blotting. Incubation of the same amount of lysate with rabbit IgG was used as a negative control. A representative of at least three experiments is shown. IP, immunoprecipitation; IB, immunoblot. (C, bottom) Cells transfected with GPX2 siRNA have elevated tBID. Beas-2B cells were transfected with GPX2 siRNA or control siRNA as above. tBID was determined by Western blot analysis. *P<0.05, ^#P<0.05.