Figure 4.

Preparation of a nuclear fraction. (A) Strategy to isolate nuclei. (B) The yield of nuclei. Fifty embryos were injected with [³H]thymidine (8,500 cpm/embryo). The acid-insoluble radioactivity of the homogenate and the nuclear fraction was scintillation counted. The values are means ± SEs of five independent experiments. (C) Photographs of subfractions. The homogenate, the upper layer, and the nuclear fraction were observed and photographed under phase-contrast or UV illumination. (Bar = 0.1 mm.) (D) Western blotting of subfractions. Each embryo was injected with HA-HA Smad2 mRNA (1 ng) and with [³H]thymidine (8,500 cpm). The homogenate (10 μg of total protein), the upper layer (10 μg of total protein), equivalent to about one-third of an animal cap, and the nuclear fraction containing acid-insoluble 1,000 cpm of [³H]thymidine, equivalent to about two animal caps, were subjected to SDS/PAGE, followed by Western blotting with the anti-HA or anti-α-tubulin antibodies. The results shown in C and D are representative of three independent experiments.