Mmp 1

The proteins of hPDLCs were harvested with a RIPA lysis buffer (Beyotime, China), and the protein concentration was measured using a BCA protein assay kit (Solarbio, China). Proteins were separated on SDS–PAGE (8–20% gel) and transferred to PVDF membranes. The membranes were blocked for 0.5 h with a blocking reagent, and then were washed and incubated with a primary antibody at 4 °C overnight. After several washes, the membranes were incubated with secondary antibodies (Beyotime, China) at room temperature for 1 h. ECL solution A and solution B (Beyotime, China) were mixed to image the membranes in a darkroom (Bio-Rad ChmiDoc, USA). The primary antibodies were listed in the following manner: GAPDH (ABclonal,1:5000,USA), NFATC1 (ABclonal,1:1000,USA), TIMP-1 (ABclonal,1:1000,USA),TIMP-2 (ABclonal,1:1000,USA), MMP-1 (ABclonal,1:1000,USA),MMP-2 (ABclonal,1:1000,USA), Runx2 (ABclonal,1:1000,USA),MMP-9 (ABclonal,1:1000,USA), COL-I (ABclonal,1:1000,USA), COL-III (ABclonal,1:1000,USA), c-Fos (CST, 1:1000, US), OCN (CST, 1:1000), CTSK (Abcam,1:1000).

The zebrafish larvae and HSF cells were extracted and centrifuged in EP tube at 12,000 rpm for 20 min, with supplement of protease inhibitors (Roche, Indianapolis, IN). The protein concentration was determined by BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). About, 30–40 μg of protein was loaded onto 10% or 12% SDS-PAGE gel and was transferred to PVDF membranes (Millipore, Billerica, MA). The proteins, including β-actin (ABclonal Biotechnology, Wuhan, China, MW42), phospho-p38 MAPK, p38 MAPK (ABclonal Biotechnology, Wuhan, China, MW41), c-jun (Cell signaling Technology, MW43), phospho-c-jun (Cell signaling Technology, MW48), NF-κB (ABclonal Biotechnology, Wuhan, China, MW65), MMP1 (ABclonal Biotechnology, Wuhan, China, MW62), IL-6 (Affinity Biosciences, Nanjing, China, MW24), IL-1α (ABclonal Biotechnology, Wuhan, China, MW20), and TNF-α (Affinity Biosciences, Nanjing, China, MW17) proteins were incubated overnight at 4°C. The next day, anti-rabbit alkaline phosphatase-conjugated antibody (KPL, Los Angeles, USA) was used as a secondary antibody for 1 hr at 30°C. Protein signals were detected via ECL method [18 (link), 19 (link)].