Rabbit polyclonal anti gapdh

Specimens were lysed in a buffer supplemented with a protease inhibitor cocktail (Roche) [14 (link)]. Concentrations of proteins were measured with the Bradford assay (Bio-Rad Laboratories, Richmond, CA, USA) using a spectrophotometer. Proteins (20 μg) were separated by SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, Billerica, MA, USA). The following primary antibodies were incubated overnight with the blocked membranes: rabbit polyclonal anti-COL2A1, rabbit polyclonal anti-MMP-13 (Santa Cruz Biotechnology), goat polyclonal anti-BMAL1, goat polyclonal anti-CLOCK, rabbit polyclonal anti-OPG, goat polyclonal anti-PER1, goat polyclonal anti-PER2, goat polyclonal anti-RANKL, mouse monoclonal anti-actin, rabbit polyclonal anti-GAPDH, and rabbit monoclonal anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA). Membranes were then incubated with the secondary antibody. Finally, specific bands were visualized with chemiluminescent reagents (Pierce Biotechnology, Rockford, IL, USA) and quantified using the ImageJ software.

A lentiviral vector containing the coding sequence of the visfatin gene was commercially sourced from Invitrogen (Shanghai, China). Recombinant human visfatin was purchased from Sigma-Aldrich (St. Louis, MO). Rabbit polyclonal anti-GAPDH was purchased from Cell Signaling Technology Inc. (Danvers, MA). Rabbit monoclonal anti-visfatin was purchased from Abcam (Cambridge, UK). Rabbit polyclonal antibodies to α-smooth muscle cell actin and MMP-8 were both purchased from Abcam (Cambridge, UK). Rat anti–mouse monoclonal antibody for macrophages was purchased from Abcam (Cambridge, UK). Rabbit polyclonal antibodies to phospho-NF-κB (p65) and phospho-IκBα were both purchased from Cell Signaling Technology Inc. (Danvers, MA). BAY11-7082 and SC-514, selective inhibitors of NF-κB, were both purchased from Sigma-Aldrich (St. Louis, MO). Rabbit polyclonal antibodies to smooth muscle-myosin heavy chain (SM-MHC), 22 kDa smooth muscle protein (SM22α), smooth muscle calponin (SM-Calponin), smooth muscle myosin light chain kinase (SM-MLCK), h-Caldesmon (h-CALD), Ki-67 and Osteopontin were all purchased from Bioss (Beijing, China). Rabbit polyclonal antibodies to MMP-1, MMP-2 and MMP-9 were also purchased from Bioss (Beijing, China).