Collagen type 1 from rat tail tendon

H. Molitrix skin was supplied by Hubei Zhongke Agriculture Co., Ltd. (Jingzhou, China). Adenosine diphosphate (ADP), trypsin -EDTA solution was purchased from Solarbio (Beijing, China). Alcalase and Protamex^® were purchased from Novozymes (Beijing, China). Collagen type I from rat tail tendon, pancreatin from porcine, and pepsin from porcine gastric mucosa were purchased from Sigma-Aldrich (Shanghai, China). Phosphate buffer solution (PBS) was purchased from HyClone (Beijing, China). Dulbecco modified Eagle’s minimal essential medium (DMEM), Fetal bovine serum (FBS), Hank’s buffered saline solution (HBSS), nonessential amino acids (NEAA), penicillin, and streptomycin were purchased from Gibco (Life Technologies, Grand Island, NY, USA). Sodium hydroxide (NaOH), trisodium citrate (Na₃C₆H₅O₇·2H₂O), hydrochloric acid (HCl), sodium bicarbonate (NaHCO₃), and other chemicals were all analytically pure grade and purchased from Sinopharm chemical reagent Co., LTD (Beijing, China).

Silver carp skin was supplied by Hubei Zhongke Agriculture Co., Ltd. (Jingzhou, China). Alcalase, papain and trypsin were purchased from Novozymes (Beijing, China). Collagen type I from rat tail tendon, pepsin from porcine gastric mucosa and pancreatin from porcine were purchased from Sigma‐Aldrich (Shanghai, China). Foetal bovine serum (FBS), Dulbecco modified Eagle's minimal essential medium (DMEM), penicillin, streptomycin, non‐essential amino acids (NEAA) and Hank's buffered saline solution (HBSS) were purchased from Gibco (Life Technologies, Grand Island, NY, USA). The BCA protein assay kit was purchased from Beijing Solarbio Science and Technology Co., ltd. (Beijing, China). Mouse cytokine array kits were obtained from R&D systems (Minneapolis, MN, USA). Commercial kits used for determination of TGF‐β₁, VEGF, PF4, GMP‐140, β‐thromboglobulin (β‐TG) and serotonin (5‐HT) were purchased from Jiancheng Institute of Biotechnology (Nanjing, China). The mouse antibodies against TGF‐β₁, type I collagen and GAPDH were purchased from Abcam (Cambridge, UK). Other chemicals used were analytical grade or better.

BR, the bovine serum albumin (BSA), rat TNF-α, LPS from Escherichia coli 0114:B4, human insulin solution, Williams’ E Medium, Collagen type I from rat tail tendon, 2,6-di-tert-butyl-4-methylphenol (BHT), Thiazolyl Blue Tetrazolium Bromide (MTT), RNAlater, and tetrabutyl-ammonium hydroxide (TBA, 40 % in water) were purchased from Sigma-Aldrich (St. Louis, MO, USA); the chloroform (HPLC grade), methanol (HPLC grade), n-hexane, ethyl acetate, and acetonitrile were purchased from Merck (Darmstadt, Germany); and 4×-Laemmli sample buffer was from Bio-Rad (Hercules, CA, USA).
As described earlier, the BR was purified before use [40 (link)]. For the experiments, BR (2.8 mg) was dissolved in 2 mL of 0.1 M NaOH and immediately mixed with 1 mL of 0.1 M phosphoric acid. The mixture was diluted with a BSA solution (660 µM BSA in 25 mM phosphate buffer, pH: 7.7) to reach a final concentration of 480 µM BR in a phosphate buffer and then serially diluted with a BSA solution to yield solutions with final BR concentrations within the range of 10–40 µM.