Figure 4.

RNase protection assay for SREBP-1a and SREBP-1c mRNAs from livers of nonfasted mice (lanes 1 and 4), mice fasted for 24 h (lanes 2 and 5), or mice fasted for 24 h and refed a high carbohydrate/low fat diet for 12 h (lanes 3 and 6). The animals are described in Table 1. Pooled total RNA was isolated from five mice in each treatment group, and a 15-μg aliquot was hybridized to ³²P-labeled cRNA probes for mouse SREBP-1a, SREBP-1c, and β-actin for 10 min at 68°C. After RNase digestion, the protected fragments were separated by gel electrophoresis and exposed to film with an intensifying screen for 8 h at −80°C. The gels were also quantified in a PhosphorImager as described in the text. The fold change in each mRNA relative to that of the nonfasted mice was calculated after correction for loading differences, using β-actin mRNA as a loading control.