At day one after birth, adipose tissue was collected from the pups that were culled to standardize the litter size. Newborns were decapitated and inguinal (subcutaneous) adipose tissue was pooled from 4 males per litter. At 9 months of age, retroperitoneal (visceral) adipose tissue, which is associated with obesity and insulin resistance21 (link),22 (link) was collected and samples frozen in liquid nitrogen and stored at −80°C till protein analysis. To control for litter effects in the adult studies, one male was studied from each of the litters. Thus when n = 6, it represents one male from each of 6 different litters in Control, IUGR and Mat-OB groups.
Protein was extracted in radioimmuno precipitation assay (RIPA) buffer that contained protease inhibitors (HALT cocktail, Pierce). Supernatant protein concentration was determined by BCA solution (PIERCE, Rockford, IL). Protein expression was determined by Western Blotting, as previously described.15 (link) Antibodies were obtained from Santa Cruz, CA unless otherwise specified and the band density was analyzed as indicated: PPARγ (57 kDa; Thermo Scientific, Rockford, IL), C/EBPα (42 kDa), SREBP1c (125 kDa), FAS (270 kDa), hormone sensitive lipase (84 kDa), SIRT1 (120 kDa), NCoR (270 kDa), SMRT (160 kDa), SRC1 (160 kDa), TIF2 (158 kDa) and β-actin (Sigma, A-5441; 40 kDa).
Protein was extracted in radioimmuno precipitation assay (RIPA) buffer that contained protease inhibitors (HALT cocktail, Pierce). Supernatant protein concentration was determined by BCA solution (PIERCE, Rockford, IL). Protein expression was determined by Western Blotting, as previously described.15 (link) Antibodies were obtained from Santa Cruz, CA unless otherwise specified and the band density was analyzed as indicated: PPARγ (57 kDa; Thermo Scientific, Rockford, IL), C/EBPα (42 kDa), SREBP1c (125 kDa), FAS (270 kDa), hormone sensitive lipase (84 kDa), SIRT1 (120 kDa), NCoR (270 kDa), SMRT (160 kDa), SRC1 (160 kDa), TIF2 (158 kDa) and β-actin (Sigma, A-5441; 40 kDa).
