FIG. 3.
Differential accumulation of MyoD protein by ectopic expression of Ckis. (A) Immunoblot showing exogenous MyoD and Ckis in transiently transfected C3H10T1/2 cells. As for Fig. 2, C3H10T1/2 cells were transiently transfected with 0.5 μg of expression vector encoding HA-MyoD (lanes 2 to 14) plus 0.5, 1, or 1.5 μg of empty expression vector (lanes 3 to 5) or 0.5, 1, or 1.5 μg of pCMV-p16Ink4a (lanes 6 to 8), pCMV-p27Kip1 (lanes 9 to 11), or pCMV-p57Kip2 (lanes 12 to 14). Whole-cell lysates (10 μg) were separated by SDS-PAGE. Proteins were transferred to nitrocellulose and immunoblotted with monoclonal antibody 12CA5 (Boehringer Mannheim) and visualized by ECL (Amersham). (B) Fifty-microgram aliquots of the same lysates from transfected cells were analyzed for expression of exogenous p16Ink4a, p27Kip1, and p57Kip2 by Western blotting with an anti-p16Ink4a (M-156; Santa Cruz), anti-p27Kip1 (F8; Santa Cruz), or anti-p57Kip2 (E-17; Santa Cruz) polyclonal antibody.