Sirt1 07 131

Immunoblot analysis was performed essentially as described previously.17, 19, 23 Briefly, total protein extract was prepared from tissues with SDS‐containing sample buffer, and the protein concentration of each sample was measured with a Detergent Compatible Protein Assay Kit (Bio‐Rad, Hercules, CA) using BSA as the standard. Equal amounts of protein extract from tissue samples were fractionated on a 5% to 20% polyacrylamide gel (ATTO Technology, Inc., Amherst, NY), then transferred to a PVDF membrane using the iBlot Dry Blotting System (Invitrogen, Carlsbad, CA). Membranes were blocked for 1 hour at room temperature with PBS containing 5% skim milk powder, and probed overnight at 4°C with specific primary antibodies (SIRT1, 07‐131; Millipore; Sirtuin3 [SIRT3], sc‐99143; Santa Cruz Biotechnology, Santa Cruz, CA; nicotinamide phosphoribosyltransferase [Nampt], sc‐67020; Abcam, Cambridge, MA; 4‐HNE, MHN‐100P; JaICA). Membranes were washed and further incubated with secondary antibodies for 15 minutes at room temperature. Sites of the antibody‐antigen reaction were visualized by enhanced chemiluminescence substrate (GE Healthcare, Little Chalfont, UK). The β‐actin band recognized by a specific antibody (A5441; Sigma‐Aldrich, St. Louis, MO) was used as a loading control. Images were analyzed quantitatively using a Fuji LAS3000 Image Analyzer (FujiFilm, Tokyo, Japan).