Western Blot Analysis

Whole cell extracts were prepared with RIPA lysis buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% sodium deoxycholate, 50 mM Tris [pH 8.0]) containing 1 mM NaF, 1 mM Na₃VO₄, and protease inhibitors. Proteins were quantified using the Bradford assay reagent (Bio-Rad) according to the manufacturer’s instructions. Proteins (20– 40 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore, USA). Membranes were blocked with 5% nonfat milk and incubated with the following antibodies: Galectin-9 (ab194338; 1:1000 dilution; Abcam), TLR 9 (ab134368, 1:1000dilution; Abcam), and GAPDH (#2118, 1:10,000; Cell Signaling). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies and visualized with Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore).