Figure 5

Reduced translation in the liver of fed Maf1^−/− mice. (A) Polysome profiles of liver samples from three WT (blue curves) and three Maf1^−/− (orange curves) refed mice. (B) Anti-puromycin immunocytochemistry of mouse liver. Mice were injected intraperitoneally with puromycin, livers were harvested after 30 min. and embedded in paraffin. Paraffin sections were incubated with an anti-puromycin antibody for detection of newly synthesized proteins (red signal). Nuclei were stained with DAPI (blue signal). The upper and lower panels on the right show negative controls from sham-injected mice. (C) Immunoblot of newly synthesized proteins in liver extracts from puromycin-injected mice. Samples were loaded according to DNA content and the blot was probed with an anti-puromycin antibody. (D) Quantification of the immunoblot in panel (C). The signal obtained in each lane was normalized to total γ-tubulin, determined by re-probing the blot with an anti-γ-tubulin antibody. (Note that the difference was larger when the data were normalized for DNA content versus γ-tubulin). (E) Liver protein content normalized to DNA content. Protein and DNA were extracted and quantified from the same lysate for each of three WT and three Maf1^−/− livers. The ratios were plotted. (F) Protein content of mouse embryonic fibroblasts normalized to cell number. Proteins were extracted and quantified from three WT and three Maf1^−/− samples, each containing one million cells. Panels (D,E) report means and standard deviations.