Rabbit polyclonal antibody against gapdh

This study was approved by the Animal Ethics Committee of Xuzhou Medical University. SD rats (1-3 days old) were purchased from the Laboratory Animal Center (Xuzhou Medical University). The animals used in this study received humane care and handling. RSV and type I collagenase were obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human TGF-β1 was purchased from Peprotech (Suzhou, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies (Grand Island, NY, USA). Cell Counting Kit-8 (CCK-8), 0.25% trypsin, dimethyl sulfoxide (DMSO), RIPA lysis buffer, phenylmethylsulfonyl fluoride (PMSF), and the bicinchoninic acid (BCA) protein assay kit were purchased from Beyotime (Guangzhou, China). The EdU (5-ethynyl-2-deoxyuridine) staining kit was purchased from RiboBio (Guangzhou, China). The hydroxyproline assay kit was purchased from Jiancheng Bioengineering Institute (Nanjing, China). A polymerase chain reaction (PCR) kit was purchased from Tiangen (Beijing, China). The mouse monoclonal antibody against Smad7 was purchased from Santa Cruz Biotechnology, Inc. (Paso Robles, CA, USA). The rabbit polyclonal antibody against GAPDH was purchased from Proteintech Group, Inc. (Rosemont, IL 60018, USA). Secondary antibodies conjugated to HRP were purchased from Zhongshan Jinqiao Biotechnology (Beijing, China).

Protein samples were extracted from the harvested cells or conditioned media, and western blotting was performed according to standard methods. After lysis with RIPA buffer containing 1% protease inhibitors (PMSF), the CNE-2 and CNE-2R cells were harvested and centrifuged. Conditioned media (CM) of CNE-2 and CNE-2R cells were treated with a protease inhibitor tablet (Roche Applied Science, Mannheim, Germany) and concentrated to an approximate volume of 200 μL using a 10 kDa molecular-weight-cut-off Amicon Ultra-15 centrifugal filter device (Millipore, Bedford, MA, USA) following the manufacturer’s instructions. Proteins extracted from the harvested cells and conditioned media were electrophoretically separated on 10% SDS-PAGE gels before being blotted onto PVDF membranes (Thermo Scientific, Waltham, MA, USA). After being blocked with 5% non-fat milk in TBST, the membranes were incubated with a rabbit polyclonal antibody against QSOX1 (1:1,000 dilution, Abcam, USA) and a rabbit polyclonal antibody against GAPDH (1:1,500 dilution; Proteintech, Chicago, IL, USA) at 4°C overnight. GAPDH served as the loading control. Then, the membranes were incubated with a goat anti-rabbit secondary antibody (1:1,500) for 1 h at room temperature. Visualization was performed with an infrared fluorescence imaging system (Odyssey; Li-Cor Co., Lincoln, NE, USA).