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. 2016 Oct 4;7(45):73558–73572. doi: 10.18632/oncotarget.12434

Figure 3. Subcellular localization of CCND1 mutants.

Figure 3

(A) Cytosolic and nuclear extracts were prepared as described in Materials and Methods from UPN-1 cells that stably expressed WT or Y44D CCND1. The extracts (30 μg per lane) were immunoblotted with indicated antibodies. β-ACTIN or GAPDH and histone H3 were used to confirm cytosolic and nuclear fractions, respectively. Numbers below the blots are relative densitometric values of corresponding bands after normalization to β-ACTIN (for WCL and Cytosolic) or histone H3 (for Nuclear) loading controls. WCL, whole cell lysates; Endo., endogenous. (B) Subcellular distribution of WT and mutant CCND1-HA proteins, which were transiently expressed in HEK-293T cells, was evaluated by immunofluorescence. Shown are representative confocal immunofluorescence images of WT and mutant CCND1-expressing cells stained with anti-HA antibody (red) followed by nuclear staining with DAPI (blue). Scale bars, 20 μm. Bar graphs show the percentages of HA positive cells that have HA staining in the nucleus from two separate experiments. Error bars, SEM; ****P < 0.0001 by a two-tailed Student's T-test. Approximately 150 HA positive cells from each group were counted.