2.7. Western blotting

Renal tissues were homogenized with ice-cold RIPA buffer (50 mM Tris (pH7.4), 150 mM NaCl, 2 mM EGTA, 2 mM Na3VO₄, 1 mM phenylmethane sulfonyl fluoride) (Beyotime, Shanghai, China) containing protease/phosphatase inhibitor cocktail (Sangon, Shanghai, China) and kept on ice for 60 min. The lysates were centrifuged at 4 ℃ for 5 min at 10 000 rpm. The protein concentration of the supernatant was determined using a BCA kit (Thermo Scientific Pierce, Rockford, IL, USA). Protein (40 μg) were separated by electrophoresis through 10% or 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels and then transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were probed with primary antibodies against TLR4 (19811-1-AP, Proteintech, Wuhan, China), MyD88 (A0980, Abclonal, Wuhan, China), IκBα (A19714, Abclonal, Wuhan, China), p65/NF-κB (66535-1-Ig, Proteintech, Wuhan, China), MAP3K7 (an isoform of MAP3K, 12330-2-AP, Proteintech, Wuhan, China), and caspase-8 (13423-1-AP, Proteintech, Wuhan, China) at 4 ℃ overnight after blocking with 5% nonfat milk in phosphate buffer saline containing 1% Tween-20 (PBST) for 1 h at room temperature. After washing with PBST for 3 times, the membranes were probed with respective secondary antibodies for 2 h at room temperature and finally developed using an ECL detection reagent. The antibody-antigen complexes were visualized by the FluorChem™ E System (ProteinSimple, San Diego, CA, USA), and the densitometry of the bands were quantified using ImageJ software (NIH, Bethesda, MD, USA).