Time after hypoosmotic stress for Focal adhesion kinase (FAK) phosphorylation to occur

Value 2 min
Organism Human Homo sapiens
Reference Kim RD et al, Activator protein 1 activation following hypoosmotic stress in HepG2 cells is actin cytoskeleton dependent. J Surg Res. 2001 Oct100(2):176-82PubMed ID11592789
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.
Comments Researchers hypothesized that hypoosmotic stress-induced activation of PKB and AP-1 in HepG2 cells is dependent on an intact actin cytoskeleton and subsequent FAK phosphorylation
Entered by Uri M
ID 104611