FIGURE 2.

Phosphorylation and degradation of Acheron. (A) ISM Western blot with an anti-phospho-serine/threonine antibody detects a protein doublet the same size as Acheron at the time of eclosion. Comparable bands were not detected before or after this time point. (B) In vitro phosphorylation and degradation of Acheron. Endogenous Acheron is present in day 18 ISM extracts but not in extracts from post-eclosion (PE) muscles. Mixing the two extracts failed to induce Acheron degradation. The addition of cGMP and IBMX resulted in a higher molecular weight Acheron protein that was then susceptible to degradation when subsequently exposed to PE ISM extracts. (C) This experiment was repeated in the presence of ³²P and then analyzed by film autoradiography, which demonstrated that Acheron is phosphorylated prior to degradation. These experiments were performed four times.