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. 2002 May;22(9):2893–2905. doi: 10.1128/MCB.22.9.2893-2905.2002

FIG. 6.

FIG. 6.

p204 can overcome the inhibition of the MyoD-, E47-, or MyoD-E47 heterodimer-dependent transcription of a reporter gene by the Id proteins. (A) Experiments with a 10T1/2 line. One microgram of p4RCAT reporter construct was transfected into 10T1/2 cells together with 1 μg of pSVGal internal control plasmid and 1 μg each of the expression plasmid(s) indicated: pCMV, MyoD, Id2, Id1, Id3, or p204 (standing for pCSA-MyoD, pFlag-Id2, pFlag-Id1, pFlag-Id3, and pCMV-204, respectively). At 48 h after transfection, the cultures were harvested and lysed, and the β-galactosidase and CAT activities were determined. The CAT activities were normalized to the β-galactosidase activities. This normalized activity was taken as 1 in the case of cells transfected with the pCMV vector. The standard deviations based on three experiments are indicated. (B) Experiments with a 10T1/2 line. One microgram of p4RCAT reporter construct was transfected into 10T1/2 cells together with 1 μg of pSVGal internal control plasmid and 1 μg each of the expression plasmid(s) indicated: pCMV, E47, MyoD, Id2, and p204 (standing for pCMV-E47, pCSA-MyoD, pFlag-Id2, and pCMV-204, respectively). However, in the case of cotransfection with E47 and MyoD, 0.5 μg of each plasmid was used. At 48 h after transfection, the cultures were processed as described for panel A. The standard deviations based on three experiments are indicated. (C) Experiments with C2C12 cell lines. The same four cell lines were used as in Fig. 5C. One microgram of p4RCAT reporter construct was cotransfected with 1 μg of pSVGal internal control plasmid into each of the stable lines: C2C12 transfected with the Flag vector, C2C12 transfected with the Flag-Id2 plasmid, con./Id2, and ind.p204/Id2. Two dishes were used for each of the con./Id2 and ind.p204/Id2 cultures. One of these two dishes was incubated without Muristerone (−Mur), and the other was incubated with 2.5 μM Muristerone (+Mur). The cultures were incubated in GM for 48 h and processed as described for panel A. The normalized activity in cells transfected with the Flag vector was taken as 1. The standard deviations based on three experiments are indicated. For further details, see Materials and Methods.