Fig. 6. Synthesis of ·NO in mouse C2C12 myotubes treated with LPS/IFN- when incubated with antisense phosphorothioates (ASP) of cav-1, cav-2 and cav-3 (A), and abrogation of caveolin-1 downregulation by protein kinase inhibitors (B). C2C12 myotubes were treated for 8 hours with antisense phosphorothioate oligonucleotides complementary to the first 21 bases of the mRNA encoding cav-1, cav-2 and cav-3. After the treatment, the muscle cells were challenged with LPS/IFN- for 36 hours and the amount of ·NO that accumulates was determined with the Griess assay. The antisense oligonucleotides (ASP) were added individually (cav-1, cav-2 and cav-3) or in combination (cav-123). A scrambled oligo corresponding to the cav-1 base sequence was also used as a control. The absence of changes in NOS2 and ß-tubulin in each case is confirmed by immunodetection (A, bottom). The results shown are representative of ten individual experiments. In order to determine the pathway of downregulation followed by cav-1 and cav-2 in the presence of the LPS/IFN- mixture, the cells were incubated with various protein kinase inhibitors for 48 hours (B). Subsequently, the levels of cav-1 were determined by immunodetection. The concentration of nitrites in the supernatant in the presence of 10 µM Erk inhibitor PD was also determined (right). ^*P<0.001 vs the LPS/IFN- condition. Error bars represent deviation from the average.