Table 2.
Reduction of BrdU incorporation by MKK6EE in MyoD-converted fibroblasts in the presence of mitogens
| Plasmids
|
% BrdU positive experiment
|
|
|---|---|---|
| 1
|
2
|
|
| Vector (GFP) | 39 | 42 |
| MKK6EE | 32 | 34 |
| MyoD | 28 | 29 |
| MyoD + SB | 32 | 35 |
| MyoD + MKK6EE | 7 | 9 |
| MyoD + MKK6EE + SB | 33 | 32 |
| MyoD + MKK6EE + PD | 5 | 11 |
Mouse 10T1/2 fibroblasts cultured in GM were transfected with the indicated plasmids. Eighteen hours after transfection, when cells were almost completely confluent, BrdU (10 μm) was added for an additional 12 hr and the incorporation of BrdU was determined by indirect immunofluorescence after fixation, using anti-BrdU antibody. Transfected cells were visualized either by staining with anti-MyoD (monoclonal anti-MyoD 5.8) or by the green fluorescent protein (GFP). The percentage of BrdU/MyoD (or BrdU/GFP) double-positive cells among 100 MyoD-positive cells was calculated.