Anti rabbit or anti mouse peroxidase labeled secondary antibody

To examine the effect of NR on p-AMPK in the glaucomatous optic nerve, optic nerve specimens (4 mm proximal lengths) were homogenized in protein extraction buffer 1 week after laser irradiation. To examine the effect of NR on p-AMPK at short time periods, optic nerve samples were collected 6, 24, and 48 hrs after one-time NR administration. One optic nerve preparation included two optic nerves. Homogenized samples were centrifuged at 15,000× g for 15 min at 4 ℃. Protein concentrations were determined with the supernatants. Each sample (3 μg) was subjected to the mini gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to an enhanced chemiluminescent membrane (EMD Millipore Corporation, Temecula, CA, USA). The membranes were blocked with 5% skim milk with tris-buffered saline (TBS) containing Tween-20 and reacted with anti-p-AMPK antibody (1:200; Sigma-Aldrich, St. Louis, MO, USA), anti-AMPK antibody (1:200; Sigma-Aldrich), or anti-β-actin antibody (1:5000; Sigma-Aldrich). After washing three times, the membranes were reacted with anti-rabbit or anti-mouse peroxidase-labeled secondary antibody (1:5000; MP Biochemicals, Solo, OH, USA). Immunoblotting was visualized with a chemiluminescence detection system (ECL Plus Western Blotting Detection Re000000agents, Amersham Pharmacia Biotech, Buckinghamshire, UK).

Optic nerve specimens (4-mm lengths) were gathered and homogenized in protein extraction buffer 1 week after injection. Homogenized samples were then centrifuged at 15,000×g for 15 min at 4 °C. Protein concentrations were determined with the supernatants. Each sample (3 μg) was applied and subjected to the mini gel (Bio-Rad Laboratories) and transferred to enhanced chemiluminescent membrane (EMD Millipore Corporation, Temecula, CA). The membranes were blocked with 5% skim milk with tris buffered saline (TBS) containing Tween-20 and reacted with anti-p62 antibody (MBL Life Science, Nagoya, Japan), anti-LC3 antibody (MBL Life Science), anti-SIRT1 antibody (Santa Cruz Biotechnology), anti-NRK1 antibody (Lifespan Biosciences Inc. Seattle, WA) or anti-β-actin antibody (Sigma-Aldrich). After three times washing, the membranes were reacted with anti-rabbit or anti-mouse peroxidase-labeled secondary antibody (MP Biochemicals, Solo, OH). Immunoblotting was visualized with a chemiluminescence detection system (ECL Plus Western Blotting Detection Reagents, Amersham Pharmacia Biotech).