| Method |
HepG2 cells were incubated for 1 h with or without 20 microM cytochalasin D, an actin disrupter, and were then exposed for up to 30 min to hypoosmotic medium (200 mOsm/L) to induce swelling. Tumor necrosis factor alpha (1.4 nM) and medium alone served as positive and negative controls, respectively. Western blots measured cytoplasmic phosphorylated or total FAK and PKB. For the detection of PKB, rabbit polyclonal IgGs specific for either total PKB or phosphorylated PKB (Ser473) (New England Biolabs, Beverly MA) were used. Protein extracts from PDGF-treated NIH 3T3 cells served as a control for the phosphorylated PKB (New England Biolabs) antibody binding. All experiments were performed in triplicate. |