TABLE 3.
Localization of IκBα proteins in β-galactosidase-positive cells following TNF-α and CHX treatment
| IκBα protein and treatment or time posttreatment (min)a | Nb | N/Cc | Cd | No MAD3e |
|---|---|---|---|---|
| myc-MAD3 | ||||
| No treatment | 28 | 40 | 4 | 28 |
| 0 | 8 | 8 | <4 | 84 |
| 15 | 28 | 20 | <4 | 52 |
| 30 | 36 | 24 | 4 | 36 |
| 60 | 16 | 52 | 4 | 28 |
| myc-MAD3-110A3 | ||||
| No treatment | <4 | 4 | 84 | 12 |
| 0 | 4 | 4 | 12 | 80 |
| 15 | 4 | 4 | 44 | 48 |
| 30 | <4 | 4 | 68 | 28 |
| 60 | <4 | 8 | 72 | 20 |
COS-1 cells were cotransfected with CMV-derived expression vectors encoding β-galactosidase and either epitope-tagged myc-MAD3 or epitope-tagged myc-MAD3-110A3. At 36 h posttransfection, the transfected cells were either refed with complete medium (No treatment) or were cultured in complete medium containing TNF-α (10 ng/ml) and CHX (100 μg/ml) for 4 h. Following the 4-h treatment with TNF-α and CHX, the transfected cells were washed and chased in complete medium for either 0, 15, 30, or 60 min to allow protein synthesis. The cellular localization of the ectopically expressed proteins was determined by double-label indirect immunofluorescence using anti-myc IgG and anti-β-galactosidase sera. At least 25 cells that were positive for coexpression of the respective myc-MAD3 and the β-galactosidase proteins were examined.
The percentage of doubly labeled cells that displayed predominantly nuclear staining of the respective myc-MAD3 protein is expressed relative to the total number of cells that displayed staining for β-galactosidase.
The percentage of doubly labeled cells that displayed predominantly whole-cell staining of the respective myc-MAD3 protein is expressed relative to the total number of cells that displayed staining for β-galactosidase.
The percentage of doubly labeled cells that displayed predominantly cytoplasmic staining of the respective myc-MAD3 protein is expressed relative to the total number of cells that displayed staining for β-galactosidase.
The percentage of β-galactosidase cells in which staining of the respective myc-MAD3 protein could not be detected is expressed relative to the total number of cells that displayed staining for β-galactosidase.