Anti mmp13 antibody

Bevacizumab was purchased from Genentech Inc. (San Francisco, CA, USA) and CsA (0.05% Restasis^®) was purchased from Allergan Inc. (Irvine, CA, USA). MMP-3 inhibitor VII and an MMP-13 inhibitor were purchased from Calbiochem (San Diego, CA, USA). The anti-MMP-3 antibody was purchased from Chemicon International Inc. (Temecula, CA, USA) and the anti-MMP-13 antibody was purchased from Abcam (Cambridge, MA, USA). Anti-β-actin was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). 4',6-Diamidino-2-phenylindole (DAPI) and Prolong Gold were purchased from Invitrogen (Carlsbad, CA, USA).

Western blot analysis was performed as previously described [24 (link)]. Briefly, total protein was isolated from SW1353 cells, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. Membranes were then incubated with an anti-HDAC4 (Santa Cruz), anti-RUNX2, anti-β-actin (Cell Signaling Technology), or anti-MMP13 antibody (Abcam, Cambridge, UK). β-actin was used as an internal control. Protein bands were visualized using an enhanced chemiluminescence system (GE Healthcare, Little Chalfont, UK), and band densities were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

We used RIPA buffer (Cell Signaling Technology, USA) to extract the total protein of primary chondrocytes. A total of 30 µg protein was loaded into SDS-PAGE gels and subsequently transferred into PVDF membranes (Beyotime). Those membranes were incubated using 5% skim milk (Beyotime) and primary antibodies including anti-COL2A1 antibody (Proteintech), anti-ACAN antibody (Abcam), anti-MMP13 antibody (Abcam), anti-ADAMTS5 antibody (Abcam), and anti-GAPDH antibody (Abcam). Furthermore, these membranes were incubated using secondary antibody (goat antirabbit IgG H&L (HRP) (Abcam) and goat antimouse IgG H&L (HRP) (ab205719, 1:2000, Abcam, UK)) for 1 hour at room temperature. An enhanced chemiluminescence substrate kit (Amersham Pharmacia Biotech, Little Chalfont, UK) was applied to visualize the protein in PVDF membranes.

HaCaT cells were lysed in lysis buffer. The protein concentrations were measured by Bicinchoninic Acid (BCA) Protein Assay. Protein samples (20 µg) were separated by SDSPAGE (10%) and transferred onto polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% fat-free milk and incubated overnight at 4 °C with anti-MMP13 antibody (Abcam, Cambridge, UK). After washing, the membrane was then exposed to secondary antibodies coupled to horseradish peroxidase. Immunoreactivities were detected using ECL reagents (Biyuntian Biotechnology Co., LTD., shanghai, China). Protein bands were quantified using Image J software.

Cell were lysed in RIPA buffer (Fisher Scientific, Pittsburgh, PA) in the present of Xpert protease inhibitor cocktail (GenDEPOT, Barker, TX). Proteins were separated by 4–20% Tris-Glycine gels (Bio-rad, Hercules, CA) and transferred to a nitrocellulose membrane. The antibodies tested included the anti-cyclin D1 antibody, anti-p53 antibody, (Cell Signaling, Danvers, MA), anti-SOX9 antibody (Santa Cruz Biotechnology, Dallas, TX), anti-MMP3 antibody, anti-MMP13 antibody (Abcam, Cambridge, MA) and β-actin antibody (Sigma-Aldrich, St Louis, MO) at 4 °C overnight. After washing, the membranes were incubated with a secondary antibody (goat anti-mouse IgG-HRP; goat anti-rabbit IgG-HRP; Sigma-Aldrich, St Louis, MO) for 1 h at RT. Blots were developed using ECL (Thermo Scientific Pierce, Rockford, IL) and protein bands were obtained by exposure to LAS-4000 image detection system (Fujifilm, Tokyo, Japan).

Cells were lysed in RIPA buffer, total protein was extracted, and the protein concentration was determined with a BCA assay. A 10% SDS-PAGE gel was loaded with 20 µg of total protein, and the separated proteins were transferred by electro blotting to PVDF membranes. The membranes were blocked with 5% non-fat dry milk in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) and incubated with the primary antibody overnight at 4°C in 5% non-fat dry milk in TBST. Immunolabeling was detected using ECL reagent (Invitrogen, Carlsbad, CA, USA). The antibodies used for western blot were from the following sources: anti-Sox9 antibody (Abcam, UK; 1:1000), anti-COL-X antibody (Abcam, UK; 1:1000), anti-COL-II antibody (Abcam, UK; 1:1000), anti-MMP13 antibody (Abcam, UK; 1:500), anti-GAPDH antibody (Sigma-Aldrich, MO, USA; 1:10000), and anti-β-Actin antibody (Sigma-Aldrich, MO, USA; 1:10000).