Rabbit polyclonal primary antibodies

Phosphorylation of mTORC1, 4E-BP1, S6K1, and rpS6 was measured using western blot techniques as previously described [22 (link)]. 50 μg of protein from each sample was loaded in duplicate onto a 7.5% or 15% polyacrylamide gel (Criterion; Bio-Rad) and subjected to electrophoresis at 150 V for 70 min. Following electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad) that were then blocked in 5% non-fat dried milk. Membranes were incubated with a primary antibody overnight at 4 °C. The following rabbit polyclonal primary antibodies (Cell Signaling, Beverley, MA) were used: mTOR (Ser²⁴⁴⁸), S6K1 (Thr³⁸⁹), 4EBP1 (Thr^37/46), and ribosomal protein S6 (Ser^240/244). Blots were incubated with secondary antibody (Amersham Bioscience) washed, and then a chemiluminescent solution (ECL plus; Amersham BioSciences, Piscataway, NJ, USA) was administered. Optical density measurements were then obtained with a digital imager (Bio-Rad) so that a densitometric analysis (Quantity One software, version 4.5.2; Bio-Rad) could be performed. Following detection of the phosphorylated protein, blots were stripped of primary and secondary antibodies and then re-probed for other proteins. All data is expressed relative to the internal standardized rodent skeletal muscle control used to normalize across blots.