FIG. 8.

Effects of decreasing or increasing the level of p204 on the levels of Id1, Id2, and Id3 in myoblasts. (A) Effect of decreasing the level of p204 on the level of Id2. Two cultures from each of three C2C12 lines expressing 204 antisense RNA (p204AS lines) and from the appropriate control line were grown to 40 to 50% confluency in GM. Thereafter, one of the two cultures from each line was shifted to DM and the other was kept in GM. After 48 h the cultures were lysed. The levels of p204 in the lysates were determined by immunoblotting. To increase the sensitivity of the Id2 assay, Id2 was immunoprecipitated from aliquots of the lysates, and the levels of Id2 in the immunoprecipitates were determined by immunoblotting. Results with only one of three p204As lines are shown. The results with the other two lines were similar. (B) Effect of increasing the level of p204 on the levels of Id2, Id1, and Id3. Cultures of stable C2C12 lines in which p204 can be induced by Muristerone (ind.p204) and the appropriate control (con.) line were grown to 40% confluency, with or without Muristerone (Mur.) as indicated, during approximately 48 h. Thereafter the medium was replaced with fresh GM, with or without Muristerone, and after a 12-h incubation the cells were lysed. The levels of p204 in the lysates were determined by immunoblotting, and the levels of Id2, Id1, and Id3 were determined by immunoprecipitation followed by immunoblotting. For further details, see Materials and Methods.