FIG. 1.

Lowering the level of p204 inhibits the fusion of C2C12 myoblasts to myotubes and the formation of myosin heavy-chain protein. (A) Stable lines of C2C12 myoblasts carrying the expression vector pCMV (control) or the antisense expression plasmid pCMV204AS (p204AS) were grown in GM for 2 days and then were shifted to DM for 4 days. The cultures were observed with a phase-contrast microscope. Bar, 150 μm. (B) The control and p204AS cultures were treated as described for panel A except that they were kept in DM for only 2 days. Thereafter the cultures were fixed, blocked with goat serum, incubated with mouse antibodies against myosin heavy chain as well as secondary antibodies against mouse IgG conjugated with fluorescein isothiocyanate, and photographed using a fluorescence microscope. (C) Assay of the p204 level in stable C2C12 lines expressing pCMV (control) or pCMV204AS (p204AS). The control (lane 1) and p204AS (lane 2) cultures were grown to confluency in GM and lysed, and 40-μg protein samples from the lysates were assayed for p204 by Western blotting with anti-p204 antiserum. The relative levels of p204 protein and the p204 band are indicated. For further details, see Materials and Methods.