Quantifying TLR Pathway Proteins in Renal Tissue

Western blot was performed according to the method described previously [40 (link)] to assess TLR pathway protein expression for TLR4, MYD88, and TRIF. Briefly, renal tissue samples were mixed with RIPA buffer containing protease inhibitor; the extracted protein was measured using a Nano Drop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Thereafter, 50 µg of the total extracted protein was separated via SDS-PAGE and blotted onto PVDF membranes. PVDF membranes were blocked by incubation in TBS enclosing 3% bovine serum albumin and 0.1% Tween 20 for one hour at room temperature. PVDF membranes were washed (TBS containing 0.1% Tween 20), and incubated first with a 1:1000 dilution of the primary antibodies (TLR 4, MYD88, and TRIF) for two hours, and then with a 1:5000 dilution of the secondary antibody at room temperature. TLR4 (SC-10741), MyD88 (SC-11356), TRIF (SC-514384), reference gene (β-actin; SC-130656), goat anti-rabbit immunoglobulin (Ig) G-horseradish peroxidase (HRP) (SC-2030) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TEX, USA). The chemiluminescence produced was detected with the C-DiGit chemiluminescence scanner (LI-COR, Lincoln, NE, USA), and the band intensity was analyzed using the scanner software.