FIG. 6.

Analysis of SREBP2 mRNA translation and protein products in male germ cells. (A) Polysome analysis. Cytoplasmic RNA from mouse testis was fractionated in the presence of Mg²⁺ (HKM) or EDTA (HKE). Equal percentages of total RNA from fractions within each gradient were examined for SREBP2gc RNA by Northern analysis. RNA in lane 2 of the HKM gradient was underrepresented due to partial loss of sample. Gradient orientations are indicated below the Northern blot results, with larger polysomes localized to the denser (bottom) fractions. (B) Western analysis of SREBP2 protein in mouse and rat tissues and spermatogenic cells. (Left panel) Four micrograms (each) of microsomal protein from enriched adult mouse spermatogenic cells (G), brain (B), liver (Li), and lung (Lu) was examined. (Right panel) Analysis of nuclear extracts (5 μg) from enriched adult mouse spermatogenic cells (G), brain (B), kidney (K), and lung (Lu). (Lower panel) Five micrograms (each) of nuclear extract from enriched adult mouse and rat spermatogenic cells (G), mouse liver (Li), and rat lung (Lu) was examined. (C) Western blot of nuclear extracts (5 μg) from adult mouse spermatogenic cells (G) and sterol-depleted mouse 3T3-L1 cells (3T3). (D) Analysis of SREBP2gc protein in nuclei (Nu) and cytoplasmic (Cyt) fractions of adult mouse spermatogenic cells (5 μg per lane).