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. 2011 Oct 17;195(2):217–229. doi: 10.1083/jcb.201102142

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© 2011 Snider et al.

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Figure 5. — Differences in GAPDH aggregation and Siah1 levels in C57BL and C3H mouse livers and effect of pioglitazone on DDC-modulated GAPDH nuclear localization in C57BL hepatocytes. (A) Nuclear fractions were prepared from C3H and C57BL mouse livers (livers from control diet [n = 3] or DDC-fed mice [n = 4]; groups are separated by dotted lines) and analyzed on the same gel by SDS-PAGE followed by immunoblotting for GAPDH under reducing or nonreducing conditions. Lamin B1 was used as a loading control. Significant levels of high molecular weight nuclear GAPDH aggregates were detected only in C57BL livers after DDC exposure. (B) C57BL livers (three independent control livers/strain and four separate livers from DDC-fed mice) express significantly higher levels of Siah1 protein after DDC treatment. Coomassie stain serves as loading control. (C) Biochemical analysis on total, cytoplasmic, and nuclei-enriched fractions from C57BL hepatocytes. The hepatocytes were cultured in the presence of vehicle (DMSO), 100 µM DDC, or 3 µM pioglitazone (Pio) plus DDC for 48 h. Detergent lysates were then prepared and blotted with antibodies to GAPDH, lamin B1 (nuclear marker), β-tubulin (cytoplasmic marker), and pan-actin (loading control).