Fig. 1. Immunocomplex kinase assays and Western blots of JNK/SAPK, p38, and ERK under normoxia and hypoxia in melanoma cell line 530. Immunoprecipitated kinases were analyzed at different time points after exposure of melanoma cell line 530 to hypoxia. In vitro kinase assays were performed in kinase buffer supplemented with 5 µCi of [-32P]ATP, 0.1 mM ATP, and substrate proteins at 30°C for 15 min. JNK/SAPK, ERK, and p38 activity was assayed with GST-cJun, MBP, and 3pK (K/M) as substrates, respectively. Proteins were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes. The intensity of radioactively labeled bands was determined by phosphorimaging. Every experiment was repeated four times. Hypoxia induced up-regulation (5-fold) of JNK/SAPK activity after 3 h. Further exposure to hypoxia led to a down-regulation of JNK/SAPK.