| 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 FAK, rabbit polyclonal antibody specific for either total or phosphorylated FAK (Tyr397) (Biosource International, Camarillo CA) was used. Protein bands were visualized using a commercially available chemiluminescence kit (Bio-Rad) and autoradiographic film exposure (Eastman Kodak, Rochester, NY). Electromobility shift assay (EMSA) measured nuclear AP-1. All experiments were performed in triplicate. |