FIG. 9.

(A) Differential accumulation of endogenous MyoD protein by ectopically expressed p57^Kip2 mutants. C2C12 myoblasts (lane 1) were transiently transfected with 0.5, 1, or 1.5 μg of a pEMSV expression vehicle encoding p57^Kip2 (lanes 2 to 4), p57ΔQT (lacking amino acids 297 to 350) (lanes 5 to 7), or p57ΔCKI (lacking amino acids 1 to 105) (lanes 8 to 10) and were cultured in high-serum (20% FCS) medium. After 48 h, 50 μg of total-cell extract was analyzed by Western blotting with anti-MyoD antibodies (Santa Cruz). (B) Differential activation of MyoD function by ectopically expressed p57^Kip2 mutants. Bars represent CAT activities from whole-cell extracts of C3H10T1/2 fibroblasts which were cotransfected with plasmids encoding the MCK-CAT reporter (0.5 μg) (lanes 1 to 8), MyoD (1 μg), wild-type p57 (p57^Kip2), p57ΔCKI, and p57ΔQT (1 μg). The corresponding empty expression vector was added to normalize DNA content to 3 μg. CAT levels were determined 48 h after transfection. Protein concentration was equalized by the Bradford method. Typically, 15-μg aliquots of total cell extracts were used for the reactions. CAT activities in duplicate plates (black and white bars) are from a representative experiment.