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. 2007 Jul 23;27(19):6818–6831. doi: 10.1128/MCB.00375-07

FIG. 5.

FIG. 5.

TRB3 colocalize with C/EBPβ within the nucleus and does not prevent its localization within centromeric regions in preadipocytes. (A) Immunohistochemistry of C/EBPβ (green) and exogenous TRB3 (red) in HEK-293 cells. Cells were transfected with 4 μg of DNA, and exogenous protein localization was assessed 48 h posttransfection. Nuclear visualization is assessed with DAPI staining (blue). (B) Immunohistochemistry of endogenous C/EBPβ (green) in 3T3-L1 preadipocytes overexpressing TRB3 or not. Cells were fixed 2 days after induction. Nuclear visualization is assessed with DAPI staining (blue). The results are representative of three independent experiments. (C) C/EBPβ protein nuclear localization in 3T3-L1 preadipocytes overexpressing TRB3 or not. Nuclear and cytoplasmic extracts were prepared 24 h postinduction, and 30 μg of protein was loaded into each lane and analyzed by Western blotting. Nuclear and cytoplasmic extracts quality was determined with lamin A and SOD4 protein. The results are representative of three independent experiments. (D) C/EBP consensus binding site oligonucleotide pull-down assays of 3T3-L1 preadipocytes nuclear extracts, with or without the overexpression of TRB3. Nuclear extracts were prepared 48 h postinduction, and 250 μg was used for the oligonucleotide pull-down assays. Elutions were analyzed by Western blotting.