P iκbα

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 proteins of liver samples were extracted with the RIPA buffer (Solarbio, Beijing, China), followed by centrifuging at 12,000 g for 15 min. Subsequently, the concentration of each protein sample was detected using the BCA Protein Assay Kit (Beyotime, Jiangsu, China). Finally, the protein sample was separately mixed with loading buffer and denatured in boiling water for 5 min.
The electrophoretic separation of the prepared proteins was performed on the sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transfered to polyvinylidene fluoride (PVDF) membranes (Millipore, United States). The membranes were incubated with the primary antibodies overnight at 4°C: Akt, p-Akt, PI3K, p-PI3K, FAK, p-FAK, mTOR, p-mTOR, NF-κB p65, p-NF-κB p65 (1:1,000, Cell Signaling Technology Inc., Beverly, MA), IκBα, p-IκBα, IKKα/β, p-IKKα/β, Bcl-2, Bax, Caspase-3, and GAPDH (1:1,000, Proteintech, Chicago, IL), α-SMA, Col-I and Col-Ⅲ (1:500, Proteintech, Chicago, IL). The membranes were washed three times with TBST buffer, followed by incubation in the dark with fluorescence-labeled rabbit/mouse anti-goat IgG (Licor, United States) at 1:10,000 dilution under agitation for 1 h at room temperature. Finally, the membranes were scanned and quantified by Image Studio Lite software (LI-COR Biosciences, NE, United States).

Protein sample concentrations were measured by BCA assay kit and 20 μg/mL subjected to Western blotting (WB), following electrophoresis in 12% SDS-PAGE. WB was conducted as follows: protein samples were blocked onto an immobilon-polyvinylidine difluoride (PVDF) membrane (Immobilon, Germany) by Bio-Rad Mini-Protran electroblotting system (Bio-Rad, China). Membrane was blocked with 5% skimmed milk powder (BD, USA) for 2 h at room temperature. Membranes were probed with antibodies for GAPDH, AKT, Syk, IκB-α (Proteintech, China) phosphorylated (P)-AKT P-Syk and P- IκB-α (CST, USA). AKT primary antibodies were used at 1:3000 dilution, GAPDH primary antibodies at 1:10,000 dilution and others at 1:1000 dilution, in primary antibody dilution reagent (Beyotime, Shanghai, China). Samples were incubated overnight at 4 °C and washed 3 times with TBST (Solarbio, Beijing, China) for 10 min. Membranes were incubated with secondary antibodies at 1:3000 dilution for 1 h, including HRP-linked goat anti-mouse IgG (CST, USA), HRP-linked rabbit anti-goat IgG (CST, USA), HRP-linked goat anti-rabbit IgG (CST, USA), then washed 4 × 5 min with TBST. To strengthen the chemiluminescence, membranes were visualized by ECL (Beyotime, China).