Western Blot Analysis of Phospho-Proteins

Total proteins were extracted from chicken hypothalamus, quantified, and subjected to Western blot as we previously described (Nguyen et al., 2015 (link)). The rabbit polyclonal anti-phospho mechanistic target of rapamycin (mTOR)^Ser2481 (#2971), anti-mTOR (#2972), anti-phospho AMP-activated protein kinase alpha (AMPKα1/2)^Thr172 (#2531), anti-AMPKα1/2 (#2795), anti-HSP90 (#PA5-17610), goat polyclonal anti-HSP60 (#sc-1052), and mouse monoclonal anti-HSP70 (#MAI-91159) were used. Protein loading was assessed by immunoblotting with the use of rabbit anti-β actin (#4967). Pre-stained molecular weight marker (Precision Plus Protein Dual Color) was used as a standard (BioRad, Hercules, CA). All primary antibodies were purchased from Cell Signaling Technology (Danvers, MA), except for the anti-HSP70 and anti-HSP90 which were purchased from Pierce Thermo Scientific (Rockford, IL) and anti-HSP60 from Santa Cruz Biotechnology (Dallas, TX). The secondary antibodies were used (1:5,000) for 1 h at room temperature. The signal was visualized by enhanced chemiluminescence (ECL plus) (GE Healthcare Bio-Sciences, Buckinghamshire, UK) and captured by FluorChem M MultiFluor System (Proteinsimple, Santa Clara, CA). Image Acquisition and Analysis were performed by AlphaView software (Version 3.4.0, 1993–2011, Proteinsimple, Santa Clara, CA).