Figure 4

Determination of cyclin D1 localization by confocal microscopy. A. MCF-7 cells were allowed to attach to coverslips overnight and were then treated with or without MG132 (50 uM) for 6 h. Cells were fixed in ice cold methanol, washed and stained overnight with an FITC conjugated anti- cyclin D1 antibody, counterstained with DAPI and examined by fluorescence microscopy. B. MCF-7 cells grown on coverslips were transfected with wild type or T286A mutant GFP-cyclin D1 and incubated overnight to allow for expression of the recombinant protein. Cells were fixed in ice cold methanol, counterstained with DAPI and examined by direct fluorescence microscopy. C. MCF-7 cells were left untransfected or transfected with the indicated GFP-cyclin D1 constructs. Cell lysates were separated by 4–20 % SDS-PAGE. Immunoblot analysis was done using antibodies against Sp1, GFP and Hsp60. D. MCF-7 cells were grown on coverslips and transfected with wild type GFP-cyclin D1. Cells were fixed in ice cold methanol and examined by confocal microscopy. E. MCF-7 cells were allowed to attach to coverslips overnight and were then fixed in ice cold methanol, washed and stained overnight with an FITC conjugated anti- cyclin D1 antibody. Cells were subsequently examined by confocal microscopy.