Placental Protein Quantification by Western Blot

Western blot analysis was performed as previously described^{22 (link)}. Placental samples were lysed in RIPA buffer in the presence of protease and phosphatase inhibitor cocktail (Pierce Biotechnology, Rockford, IL). Protein concentrations were determined by BCA assay (Pierce, Rockford, IL). Thirty µg of protein lysate was reduced (Dithiothreitol, DTT), and subjected to sodium dodecyl sulfated polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. The blots were incubated with antibodies specific for Acel-K27-His3, DNMT1, DNMT3a, PGC-1α (Abcam, Cambridge, MA), TFAM, P(S97)-ACC, or β-actin (Cell Signaling Technology, Danvers, MA), and detected by enhanced chemiluminescence (Pierce, Rockford, IL). The blots were quantified and analyzed by imaging densitometry with Image Lab Software (Bio-Rad, Hercules, CA).