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

Figure 1. CCND1 mutations increase protein levels.

Figure 1

(A) Diagram shows recurring point mutations (red bars) in the N-terminus of CCND1 protein. (B) Immunoblot analysis of WT and mutant CCND1 expression. UPN-1 or Z-138 cells were transduced with WT or mutant CCND1-HA and selected for stable expression by hygromycin. Cell lysates (10 μg per lane) were separated by SDS-PAGE gel and immunoblotted with indicated antibodies. Arrow indicates a mobility shift of the CCND1-HA protein. Arrowhead indicates endogenous CCND1. Bar graphs below the immunoblot show relative densitometric values of indicated bands after normalization to GAPDH loading controls. (C) Quantitative PCR analysis of mRNA expression of WT and mutant CCND1. Cell lines generated as described in (B) and mRNAs were harvested for quantitative PCR analysis of CCND1-HA mRNA expression. Shown are the means of mRNA expression levels after normalization to GAPDH signals from four independent amplification experiments. Error bars, SD. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by a two-tailed Student's T-test. (D, E) UPN-1 or Z138 cells expressing WT or Y44D CCND1-HA were treated with 10 μM of cyclohexamide (CHX) for indicated times and 10 μg of cell lysates per lane were prepared for immunoblot analysis with indicated antibodies. Arrow, CCND1-HA protein. Arrowhead, endogenous CCND1. Bar graphs below immunoblots in (D, E) show relative densitometric values of indicated bands from the blots after normalization to time zero control samples.