Fig. 5. Association of the three caveolin isoforms to NOS2 purified and immunoprecipitated from mouse C2C12 myotubes. Separation of the total lysate of LPS/IFN--treated C2C12 myotubes into a soluble [Supernatant (Sup.)] or particulate [Pellet (Pel.)] fraction and analysis of the NOS2, cav-1, cav-2 and cav-3 distribution as judged by immunodetection (A). Additionally, NOS2 was immunoprecipitated from C2C12 myotubes treated with LPS/IFN- and the association with CaM, cav-1, cav-2 and cav-3 was determined (B). Finally, NOS2 was affinity purified using an ADP/Sepharose column (C). In this experiment, a lane was loaded with the amount of total cell lysate (Lys.) to give a similar NOS2 immunoreactivity to that of the purified sample (Pur.). Under this circumstance, the amount of cav-1, cav-2 and cav-3 that was retained bound to the purified NOS2 was compared with that present in the total lysate as determined by immunoblot (C).