T iκbα

The protein used for western blotting was extracted using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology) supplemented with protease inhibitors (Roche). The proteins were quantified using the BCA Protein Assay Kit (Pierce). The western blot system was established using a Bio-Rad Bis-Tris Gel system according to the manufacturer’s instructions.
Primary antibodies of NOD1 (ab170547; Abcam), p-IKK (#2697), t-IKK (#11930), p-IκBα (#2859), t-IκBα (#4814), p-NF-κB (#3033), t-NF-κB (#8242), p-Smad3 (#9520), t-Smad3 (#9523) and PAI-1 (#11907; Cell Signaling Technology) were prepared in 5% blocking buffer at a dilution of 1:1000. Primary antibodies were incubated with the membrane at 4 °C overnight, followed by washing and incubation with secondary antibody (1:5000, Abcam) marked by horseradish peroxidase for 1 h at room temperature.
After rinsing, the polyvinylidene difluoride (PVDF) membrane-carried blots and antibodies were transferred into the Bio-Rad ChemiDoc XRS system, and then Immobilon Western Chemiluminescent HRP Substrate (Millipore) was added to cover the membrane surface. The signals were captured and the intensity of the bands was quantified using Image Lab Software (Bio-Rad).

DEN, AAF, CCl₄, and Sb were obtained from Sigma chemicals company (St. Louis, MO, USA). Assay kits for urea, creatinine, and uric acid were purchased from Biosystem (Spain). Antibodies for p-PI3K and β-actin were obtained from Santa Cruz Biotechnology (USA). t-PI3K, t-Akt, p-Akt, t-p65, p-p65, t-IκBα, and p-IκBα antibodies were purchased from Cell Signaling Technology, Inc. (USA). Cleaved-caspase-3 antibody was obtained from Merck Millipore (Germany). Other analytical grade reagents and chemicals were purchased from local suppliers.