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  • Cultured cells or mouse lung tissues

    2020-08-05

    Cultured cells or mouse lung tissues were harvested and lysed in Radio-Immunoprecipitation Assay (RIPA) buffer (0.05 mol/l Tris–HCl pH 7.4, 0.15 mol/l NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mmol/l ethylenediamine tetraacetic SB 203580 (EDTA), 1 mmol/l phenylmethylsulfonyl fuoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin and Phospho-STOP). Protein concentration was determined by BCA protein assay kit (Pierce Biotech, Rockford, IL) and equal amounts of protein were resolved by 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). After blocking in Tris-buffered saline containing 5% skim milk and 0.1% Tween-20, membranes were incubated with primary antibodies, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibodies. The dilution of primary antibodies for immunoblot was as follows: DDR2 (1 : 1000, R&D); β-actin (1 : 500, Booster); Tublin (1 : 1000, Booster); α-SMA (1 : 500, abcam), phosphotyrosine specific monoclonal antibody 4G10 (1 : 1000, Millipore). Statistical analyses were performed using the Statistical Product and Service Solutions (SPSS) software program. One-way analyses of variance (ANOVAs) followed by least significant difference SB 203580 (LSD)-t tests were used to make comparisons between pairs of groups. Student\'s t-test was used to examine the differences between the two groups of data. A two-tailed P-value of <0.05 was considered significant.
    Supplementary Material
    Acknowledgments
    Introduction The Discoidin domain receptor was discovered by homology cloning based on catalytic kinase domain and considered as orphan receptor until 1997 when two independent groups discovered that several different collagens were ligand for DDR receptors [1,2]. A cell surface receptor tyrosine kinase, discoidin domain receptor 2 (DDR2) plays a key role in communication around the circumventing environments of extracellular matrix (ECM). The signaling role of DDR2 in the developments of bone and cartilage degradation due to deposition of ECM components has suggested the paramount contribution in patho-physiological scenario in cartilage [3]. The mechanical injuries, degradation of cartilage, inflammatory disorders and age-related changes cause accumulation of collagen at the injured site of joints. At the early stage of osteoarthritis, the expression level of DDR2 is elevated in chondrocytes because of collagen type II protein accumulation and proximity upon the degradation of cartilage. This leads to release of matrix metalloproteases (MMP). The level of MMP-1 in human fibrosarcoma cell line HT1080 has found to be overexpressed through DDR2 activation, suggesting of regulatory role in cartilage degeneration [3]. The incremented expression of DDR2 enhances the binding of receptor to collagen type II, which in turn switch on the expression of MMP-13, causing a severe damage to extracellular matrix of cartilage, as well [4,5]. Therefore, DDR2 receptor in chondrocyte is sensed by the microenvironments created due to mechanical stress or biochemical insults. Collagen type II in cartilage could trigger more DDR2-dependent collagen signaling towards the pathological condition.