Fig. 10. Hsp72 antisense oligonucleotides block the inhibitory effect of mild heat shock on JNK activation and UV-induced apoptosis. NIH 3T3 cells were transiently transfected with 10 µM Hsp72 antisense (AS) or nonsense (NS) oligonucleotides using GenePORTER 2. After 30 h of transfection, the cells were exposed to mild heat shock (HS) at 43°C for 20 min and then further incubated at 37°C for 16 h. (A) Cell lysates were subjected to immunoblot analysis with mouse anti-Hsp72 monoclonal antibody. Cell lysates were also subjected to immunoprecipitation using mouse anti-JNK1 monoclonal antibody. The immunopellets were then analyzed by immunoblotting probed with mouse anti-Hsp72 antibody. IgG_H, the heavy chain of immunoglobulin G. (B) Cells were irradiated with UV light (60 J/m²) and then incubated for 1 h. Cell lysates were then analyzed for JNK1 activity by immunocomplex kinase assay using anti-JNK1 antibody. Intracellular levels of JNK1 were determined by immunoblot analysis with anti-JNK1 antibody. (C) Cells were irradiated with UV (60 J/m²), further incubated for 16 h, and then measured for apoptotic cell death with DAPI staining as in Figure 9. Data shown above are the mean ± average deviation of triplicates from one of two representative experiments.