Anti mmp 1

Acetonitrile (HPLC grade) and glacial acetic acid (99.0 % purity) were obtained from Duksan Pure Chemicals Co. (Ansan, South Korea). High purity nitrogen gas was provided by Shinyang Oxygen Co. (Seoul, South Korea). _L-ascorbic acid was purchased from Sigma-Aldrich (St Louis, MO, USA). An MMP-1 immunoassay ELISA kit was purchased from Calbiochem Inc. (Darmstadt, Germany), and a type-1 procollagen immunoassay ELISA kit was purchased from Takara Bio Inc. (Otsu, Japan). Protease and phosphatase inhibitor cocktails were purchased from Roche (Mannheim, Germany). Western Blot was performed using following antibodies: anti-MMP-1 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), type-1 procollagen from Abnova Corporation (Taipei, Taiwan), and β-actin from Santa Cruz Biotechnology, Inc.

Western blot assays were carried out as previously described [25 (link)]. The protein from primary human chondrocytes was isolated after 24 h of treatment. The primary antibodies used were rabbit polyclonal anti-MMP1 (1:500), anti-MMP2 (1:500), anti-MMP13 (1:500), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000), goat polyclonal anti-MMP3 (1:500), mouse monoclonal anti-MMP9 (1:1000; Santa Cruz Biotechnology, Inc.), and anti-SRY-related high mobility group-box gene9 (SOX9) (1:2000; Millipore).

The Western blotting procedure is similar to our previous reports [17 (link), 23 (link)]. Briefly, the liver tissue from the rat model was lysed in RIPA buffer containing a proteinase inhibitor cocktail (Biocolor BioScience & Technology, Shanghai, China). The protein concentration was determined using bicinchoninic acid (Bioss, Beijing, China). Protein was loaded at 30 μg/gel each lane, separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated with rabbit primary antibodies anti-MMP-1 (1:1000, sc-241561) or TIMP-1 (1:1000, sc-5538) (Santa Cruz Biotech, USA) and anti-GAPDH (rabbit monoclonal antibody, 1:1000, Ab181602, Abcam, UK) at 4 °C overnight, then washed extensively with 0.1% Tween-20 in PBS and incubated with a secondary antibody conjugated to horseradish peroxidase (1:5000; sc-2004, Santa Cruz, USA) at room temperature for 3 h. The blot was visualized using the ECL system (Amersham, UK).
The serum levels of Smad7, collagen I, collagen III, laminin and hyaluronic acid were detected using ELISA according to the manufacturer’s instruction. The ELISA kits for Smad7 (# CSB-E09225r) and hyaluronic acid (# CSB-E08120r) were purchased from CUSABIO Technology LLC (Wuhan, China), and the kits for collagenase I (# CX20064), collagenase III (# kt210320) and laminin (# KT20202) were from MSKBIO (Wuhan, China).

Western blot analysis was carried out to examine the expression of proteins involved in the apoptotic pathway. To perform this assay, 75,000 cells/mL were seeded and treated with ST compounds (10 nM, 20 nM, 40 nM, 60 nM, 80 nM) for 48 h and whole cell lysate was prepared using RIPA buffer (25 mM Tris-Cl pH 7.6, 150 mM Sodium chloride, 1% NP-40, 1% Sodium deoxycholate, 1% Sodium Dodecyl Sulphate, 1 mM Phenylmethylsulphonyl fluoride, 1 mM Sodium orthovanadate). Crude cell lysates (30–40 μg) were electrophoresed on SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and were transferred on to Polyvinylidene fluoride membrane (Millipore, USA) to probe with respective antibodies. The primary antibodies against caspase 9, cleaved caspase 9, caspase 3, cleaved caspase 3, caspase 8 and Horseradish peroxidase-labeled secondary anti-rabbit antibodies were purchased from Cell Signalling Technology, Beverly, MA. Anti-tubulin, Anti-MMP1 and its secondary mouse antibody were purchased from Santa-Cruz Biotechnology, Santa Cruz, CA. The membrane was probed with appropriate antibodies and was developed using chemiluminescence reagent (Clarity Western ECL blotting substrate, Biorad). The blot image was captured by using a Syngene G: Box gel doc system. Protein band image quantification was done using GelQuant. Net, Biochem Lab solutions.

Western blot analyses were performed as previously described [18 (link)]. Briefly, cells were lysed and sonicated in RIPA buffer (Thermo Scientific, IL, USA). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoretic separation, protein bands were transferred to a Millipore Immobilon-P membrane followed by immunoblotting with antibodies against molecules of interest. The following primary antibodies were used for immunoblotting: anti-Smad2 (Cell Signaling, MA, USA, 1:1000), anti-p-Smad2 (Cell Signaling, 1:1000), anti-Smad3 (Cell Signaling, 1:1000), anti-p-Smad3 (Cell Signaling, 1:1000), anti-Akt (Cell Signaling, 1:1000), anti-p-Akt (Thr308 and Ser473) (Cell Signaling, 1:1000), anti-S6K (Cell Signaling, 1:1000), anti-p-S6K (Cell Signaling, 1:1000), anti-α-SMA (SIGMA, 1:2000), anti-fibronectin (Abcam, 1:1000), anti-vinculin (SIGMA, 1:1000), anti-paxillin (BD Bioscience, 1:1000), anti-MMP1 (Santa Cruz Biotechnology, CA, USA, 1:1000) and anti-GAPDH (Santa Cruz Biotechnology, 1:5000) antibodies. Chemiluminescence detection was performed using ECL reagent (Bio-Rad Laboratories. Inc., CA, USA). The signal intensities of bands were quantified with ImageJ software (NIH).