Anti nampt

Adv-vectors (KLF4, PDGFRA and NAMPT) were used to overexpress target genes, and sh-RNAs (KLF4, PDGFRA and NAMPT) were used to knock down target genes. All were purchased from Viogene Bio. (Jinan, Shandong, China). The KLF4 luciferase reporter plasmid (pGL4-KLF4) was purchased from Yeasen Bio. (Shanghai, China). A fluorescent dual luciferase reporting system and murine leukemia virus reverse transcriptase were obtained from Promega Co. (Mannheim, Germany). The SA-β-gal-kit was obtained from Beyotime (Haimen, China). The MitoSOX Mitochondrial Superoxide Indicator was obtained from Yeasen (Shanghai, China). The anti-p21 antibody was obtained from HUABIO (Hangzhou, China). The anti-PDGF-BB, anti-PDGFRA, anti-PDGFRB, anti-KLF4, anti-NAMPT, anti-PAI-2, anti-uPA and anti-β-actin antibodies and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Other reagents were purchased from Sigma (St. Louis, MO, USA).

Total protein extracts were prepared using RIPA buffer (Beyotime) in the presence of a proteinase inhibitor mixture (Roche Applied Science). Nuclear and cytoplasmic protein extracts were prepared using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). Protein extracted from the cells or from fresh-frozen tissues was loaded and separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes by electrophoresis and were incubated with the primary antibodies. Immunoreactive bands were detected by chemiluminescence using corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies and enhanced chemiluminescence (ECL) detection reagents. Gray intensity analysis of the western blot images was conducted using ImageJ software. Then, the relative protein abundance was determined. The primary antibodies used for western blot include the following: anti-NAMPT (Cell Signaling Technology, # 61122), anti-GAPDH (Cell Signaling Technology, # 5174), anti-β-catenin (Cell Signaling Technology, # 8480), anti-cyclin D1 (Cell Signaling Technology, # 2978), and anti-Axin (Cell Signaling Technology, # 2087).