FIG. 2.

The reduced level of Se-GPx1 mRNA in Se-deficient hepatocytes is due to a decrease in the level of cytoplasmic Se-GPx1 mRNA but not nuclear Se-GPx1 mRNA. Nuclear (N) and cytoplasmic (C) RNA was isolated (method 1 [4]) from hepatocytes of each pair of rats fed either a Se-deficient (−) or a Se-supplemented (+) diet. Se-GPx1 and β-actin RNAs in each sample were analyzed by RT-PCR. The left-most four lanes consist of serial dilutions of cytoplasmic RNA (5.0, 1.3, 0.6, and 0.3 μg, from left to right), which were used to establish that there is a linear relationship between the amount of input RNA and the amount of each RT-PCR product. Size standards for RT-PCR products of Se-GPx1 pre-mRNA and Se-GPx1 mRNA were provided by the PCR amplification of, respectively, 25 pg of pmCMV-GPx1 (Se-GPx1 gene) and 25 pg of pGPx1211 (Se-GPx1 cDNA) with the same primers that were used to amplify hepatocyte cDNA.