MAPK p38 Activity Assay

MAPK p38 activity assay was performed as described before (Huang et al., 1999). NCM460 cells were seeded at 20.000 cells/cm² in 6-well plates and grown for 3 days before treatment. For kinetic measurements, cells were treated with the CM for different periods. For evaluation of STW5 and STW5-II effects, cells were preincubated with the combination preparations for 4 h before adding the CM. Lysed cells were incubated overnight with immobilized anti-pp38 antibody beads. Kinase assays were performed by incubating the immunoprecipitates with ATF-2 fusion protein and ATP for 30 min at 30°C. The pATF-2 content was determined by western blot.