FIG 5.

Levels of the mutant CUGBP1-S302A protein are dramatically reduced in DEN-treated S302A mice. (A) Western blots with antibodies to PCNA, cyclin D1, Thy-1, and p53 using nuclear extracts of livers from WT and S302A mice 38 weeks after DEN injection. The tumor lane shows protein extracts isolated from big external tumor nodules. (B) Quantification of PCNA and p53 protein levels as ratios to β-actin. (C) Western blots of livers of WT and S302A mice (control and DEN-treated) with antibodies to CUGBP1 and Gank. The red arrow shows the position of Gank immune-reactive protein, the molecular mass of which corresponds to the sum of CUGBP1 and Gank. (D) Bar graphs show calculations of levels of CUGBP1 protein (left) and CUGBP1 mRNA (right) as ratios to β-actin. (E) CUGBP1 was immunoprecipitated from protein extracts of untreated and DEN-treated mice (shown on the top). The IPs were probed with antibodies to Gank and then reprobed with antibodies to CUGBP1. (F) Levels of Gank in CUGBP1 IPs were calculated as ratios to CUGBP1. (G) CUGBP1 was immunoprecipitated from protein extracts of livers of mice shown on the top. The IPs were probed with antibodies to Gank. Input shows Western blotting of CUGBP1 and Gank. The final lane shows CUGBP1 IP from livers of GLKO mice as a control. *, P < 0.05; **, P < 0.01.