Anti mmp 13 polyclonal antibody

Cells were washed in phosphate buffered saline (PBS) and solubilized in lysis buffer (20 mmol/L Tris‐HCl pH7.4, 150 mmol/L NaCl, 0.1% sodiumdodecyl sulphate [SDS], 1% Sodium deoxycholate, 1% Triton, 1 mg Aprotinin, 1 mg Leupeptin) for 60 min on ice. Lysates were centrifuged at 2500g for 5 min. The protein concentration was determined by means of the Bradford protein assay (Bio‐Rad Labs, Richmond, CA) using bovine serum albumin as the standard. Twenty‐five micrograms of protein was resolved by electrophoresis through 12% polyacrylamide gels, was electrotransferred to a polyvinylidene difluoride filter (Millipore, Bedford, MA), and was then blotted with the first antibody. Following washing three times and second antibody incubation, the membranes were rinsed and the bound antibodies were detected using an enhanced chemiluminescence (ECL) detection system (Amersham, Buckinghamshire, UK). The primary antibodies used in this study were anti‐PAI‐1 polyclonal antibody (1:400 dilution; ab31280 Abcam, Cambridge, UK), anti‐MMP‐13 polyclonal antibody (1:400 dilution; ab39012 Abcam, Cambridge, UK), and anti‐β‐actin (1:2000; AC‐15; Sigma). The secondary antibodies used in this study were donkey anti‐goat IgG‐HRP (Santa Cruz Biotechnology, SantaCruz, CA, USA), goat anti‐rabbit IgG‐HRP (Santa Cruz Biotechnology), and anti‐mouse IgG‐HRP (MBL, Nagoya, Japan).

Rabbit Anti MMP‐13 polyclonal antibody (Abcam, ab39012), Rabbit Anti ADAMTS‐5 polyclonal antibody (Abcam, ab41037), Rabbit Anti p16^INK4a polyclonal antibody (Thermo Fischer, PA1‐30670), Rabbit Anti LC3B polyclonal antibody (Thermo Fischer, PA1‐16930), Alexa Fluor 488 goat anti‐rabbit IgG (H+L) (Abcam, A11008), and HRP conjugated goat anti‐rabbit secondary antibody (Thermo Fischer, 31460).