*4.7. Western Blot Analysis*

Protein isolation was performed using RAW264.7 cells plated in a 60 mm culture dish at a density of 7 × <sup>10</sup><sup>5</sup> cells/well. After 24 h, RAW264.7 cells were pre-treated with 150 <sup>μ</sup><sup>M</sup> wistin for 30 min, followed by LPS (0.1 μg/mL) treatment. The protein was isolated at two time points, 2 and 24 h. Cells were harvested and lysed with lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA pH 8, 1% Triton X-100, 1% sodium deoxycholate, 30 mM NaF, 1.5 mM NaVO4, 1 mM PMSF, and 1 mg/mL each of aprotinin, leupeptin, and pepstatin A) and sonicated for 10 s. The cell lysates were centrifuged at 13,000 rpm for 10 min at 4 ◦C (Centrifuge 5424 R (rotor type: FA-45-30-11), Eppendorf, Hamburg, Germany) and quantified with the help of the Bradford (Abcam, Cambridge, UK) assay. The proteins (15 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween-20 (TBS-T) at room temperature (RT) for 30 min. Membranes were then incubated with suitable primary antibodies overnight at RT with gentle shaking. The following day, the membranes were washed with TBS-T for 30 min. Membranes were then incubated with secondary horseradish peroxidase (HRP)-conjugated anti-rabbit/mouse IgG antibodies for 1 h at RT. Finally, the membranes were detected using an ECL reagent (Cytiva, Malborough, MS, USA), and the bands were visualized using Chemidoc (iBright™ CL1500, Invitrogen). Band intensities were analyzed using ImageJ software. β-actin was used as the loading control.
