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. 2006 Mar;80(5):2257–2266. doi: 10.1128/JVI.80.5.2257-2266.2006

FIG. 1.

FIG. 1.

Downregulation of p53 protein amount by vIRF1. (A) vIRF1 expression deceases the endogenous p53 protein amount. TRExBJAB-cDNA5, TRExBJAB-vIRF1, TRExBCBL1-cDNA5, and TRExBCBL1-vIRF1 cells were treated with doxycycline (Doxy) for the indicated times, and their cell lysates were used for immunoblotting with anti-p53, anti-vIRF (myc), and antitubulin antibodies. (B) RNase protection assay. TRExBCBL1-cDNA5 and TRExBCBL1-vIRF1 cells were treated with doxycycline for the indicated times, their total RNA was isolated by phenol-chloroform extraction, and contaminated DNA was removed by DNase I treatment. Thirty micrograms of total RNA was then subjected to RPA using an RPA kit from BD PharMingen (San Diego, CA) according to the manufacturer's recommendation. (C) Reduction of p53 protein stability by vIRF1 expression. TRExBJAB-cDNA5, TRExBJAB-vIRF1, TRExBCBL1-cDNA5, and TRExBCBL1-vIRF1 cells were stimulated with doxycycline for 24 h, followed by treatment with cycloheximide (50 μg/ml) for the indicated times. Whole-cell lysates were used for immunoblotting with anti-p53 and antitubulin antibodies. The half-life of p53 was calculated by the formula described in Materials and Methods.