Mouse renal tubular epithelial cell line NRK-52E cells was obtained from the Chinese Type Culture Collection (Shanghai Institute of Cell Biology, China) and were inoculated in a sterile culture flask containing calf serum high-glucose DMEM at 37°C in a humidified atmosphere of 5% CO2. When growing together like cobblestones, the cells were seeded on 6-well BioFlex collagen-coated culture plates and grown to 80% confluence. The FBS concentration was reduced to 1% 24 hours prior to the experiments. The cells were stimulated with LPS (Sigma, St. Louis, MO) of different concentrations and duration. To study the role of Tec kinase in renal tubular epithelial cells, different concentrations of LFM-A13, a leflunomide metabolite analogue, were added to the cells 1 h before stimulation with LPS.
The reagents used in this study were purchased from the following sources: LFM-A13 from Tocris Bioscience and Tec siRNA from Genepharma Co., Ltd. (Shanghai, China); and all monoclonal antibodies for NF-κB p-p65, p65, IκBα, and p-IκBα were purchased from Protein Tech Group (Chicago, IL, USA). The antibodies for Tec protein, TLR4, MyD88, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
The reagents used in this study were purchased from the following sources: LFM-A13 from Tocris Bioscience and Tec siRNA from Genepharma Co., Ltd. (Shanghai, China); and all monoclonal antibodies for NF-κB p-p65, p65, IκBα, and p-IκBα were purchased from Protein Tech Group (Chicago, IL, USA). The antibodies for Tec protein, TLR4, MyD88, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).