Protein free t20 blocking buffer

Proteins of 30 – 50 µg per lane were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrotransferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA). After being incubated with the Protein-free T20 Blocking Buffer (Thermo Scientific, Rockford, IL), the membranes were probed overnight at 4°C with various primary antibodies: rabbit polyclonal anti-Akt antibody (catalog number: 9272, Cell Signaling Technology), rabbit polyclonal anti-phospho-Ser473-Akt antibody (catalog number: 9271, Cell Signaling Technology) and rabbit polyclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (catalog number: G9545, Sigma, St Louis, MO). The membranes were incubated with the appropriate horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies for 1 h at room temperature. The protein bands were visualized with the enhanced chemiluminescence method using a Genomic and Proteomic Gel Documentation (Gel Doc) Systems from Syngene (Frederick, MD, USA). The densities of Akt and phospho-Akt protein bands were normalized to those of GAPDH to control for errors in protein sample loading and transferring during Western blotting. The results of various experimental conditions were normalized to the corresponding data of control cells in the same experiment.

The mouse cerebral cortex was micro-dissected and placed in an immunoprecipitation assay buffer supplemented with 1% cocktail of protease and phosphatase inhibitors. The tissue was then homogenized in the buffer and centrifuged at 13,000 rpm for 30 minutes at 4 °C, after which the supernatant was collected. The protein concentration was quantified using the bicinchoninic acid method. Aliquots of protein (30–40 μg/lane) were separated by polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane. The membranes were blocked with a Protein-Free T20 Blocking Buffer (37573, Thermo Fisher Scientific) and then incubated with the rabbit polyclonal antibody to the endothelial nitric oxide synthase (eNOS) and the monoclonal antibody to the phospho-eNOS (1:1000; catalogue number 9572 and 9570, respectively, Cell Signaling Technology) at 4 °C overnight. Band volumes of the target proteins were normalized to those of non-phosphorylated protein. The band volumes of the samples in the obese group were normalized to those in the control group.