Nampt antibody

The antibodies used in this study include Nampt antibody (abcam, Cambridge, MA), FoxO1 antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA), GAPDH antibody (Sigma-Aldrich, St. Louis, MO), thioredoxin1 antibody (generated by this laboratory), Ac-FoxO1 antibody, Akt antibody, phospho-specific Akt antibody, ERK1/2 antibody, phospho-specific ERK1/2 antibody, STAT3 antibody, phospho-specific STAT3 antibody, Bcl2 antibody, Bcl-xL antibody, Bax antibody (Cell Signaling Technology, Danvers, MA), and MnSOD antibody (BD Transduction Laboratory, San Jose, CA). Ly.6B.2 antibody (AbD Serotec, Raleigh, NC)

After treatment, PC12 cells were washed twice with PBS. The cells were lysed in RIPA buffer produced by Millipore (Temecula, CA, USA) which contained 1 mM phenylmethanesulfonyl fluoride and 1% protease inhibitor cocktail produced by CWBio (Beijing, China). After centrifugation at 12,000 rpm for 10 min at 4 °C, the protein concentrations of the samples were determined by BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA). Thirty μg of the sample was electrophoresed through a 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred to 0.45-μm nitrocellulose membranes. The blots were incubated with a polyclonal Nampt antibody from Abcam (Cambridge, UK), which was diluted in TBST containing 1% bovine serum albumin at 1:2,000 ratio, at 4 °C overnight. Subsequently, a horse radish peroxidase-conjugated secondary antibody produced by Epitomics (Zhejiang Province, China) diluted by TBST containing 1% bovine serum albumin at 1:3,000 ratio was used for incubation of the blots for 1 h at room temperature. The normalization of the sample was conducted by using a Tubulin antibody produced by proteintech (Wuhan, China). Quantifications of the bands were performed by Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD, USA).