Article Figures & Data
Figures
- Fig. 1.
Identification of the menin protein. A, a rabbit polyclonal antibody was raised against the peptide shown. Total cell lysates (50 μg) were separated by SDS-PAGE, Western blotted, and probed with the anti-menin polyclonal antibody. B, left panel, GH4C1 cells transfected with (Lane 1) empty vector and (Lane 2) menin cDNA. B, right panel, COS-7 cells transfected with (Lane 1) empty vector and (Lane 2) menin cDNA. C, subcellular localization of menin. Nuclear (N), membrane (M), or cytoplasmic (C) fractions of GH4C1 cells were separated by SDS-PAGE (20 μg protein/lane) and immunoblotted. As controls for the fractionation procedure, antibodies against TFIIHp89 and β-tubulin were used for nuclear and cytoplasmic proteins, respectively.
- Fig. 2.
Cell cycle regulation of menin. A, total cell lysates (30 μg) from unsynchronized GH4C1 cells (Asynchronous) or from cells serum-stimulated for 0, 4, 8, 12, 16, 20, and 24 h (Lanes 0, 4, 8, 12, 16, 20, and 24) after serum starvation for 24 h were separated by SDS-PAGE and immunoblotted with anti-menin antibody. Results of densitometric analysis are indicated below the immunoblot. B, flow cytometry of propidium iodide-stained cells.
- Fig. 3.
Cell cycle synchronization and menin protein expression. A, GH4C1 cells were serum-starved for 24 h; cultured with complete media including 12 μm aphidicolin, 400 μm mimosine, or 1 μg/ml Colcemid for 24 h; and then released from cell cycle blockade by culture in complete media for the indicated times (h). Total cell lysates (30 μg) were examined with anti-menin antibody by Western blotting. Results of densitometric analysis are indicated below the immunoblot. B, flow cytometry of propidium iodide-stained cells. C, Western blot of nuclear extracts (20 μg) probed with anti-Rb antibody.
- Fig. 4.
A, subcellular localization of menin at different cell cycle stages. GH4C1 cells were serum-starved for 24 h and cultured with complete media including 400 μm mimosine or 1 μg/ml Colcemid for 24 h. Nuclear (N), membrane (M), or cytoplasmic (C) fractions of GH4C1 cells were separated by SDS-PAGE (20 μg protein/lane) and immunoblotted with anti-menin antibody. Jun D expression is shown at different cell cycle stages. GH4C1 cells were serum-starved for 24 h. B, cells were then stimulated with serum and harvested at the indicated times (h). C, cells were cultured with complete media including 400 μm mimosine or 1 μg/ml Colcemid for 24 h, and then the cell cycle blockade was released by culture in complete media for the indicated times (h). Nuclear extracts were prepared, 20-μg aliquots were separated by SDS-PAGE, and Western blotting was carried out with anti-Jun D antibody.
Volume 59, Issue 20
