THP-1 cells were harvested and incubated for 30 min with lysis buffer containing Tris (62.5 mM, pH 7.5), 1% Triton X-100, and 10% glycerol. The lysates were centrifuged at 14,000 × g for 10 min and supernatants were collected. Protein concentration in lysates was measured by Quickstart Bradford Dye Reagent, 1× Protein Assay kit (Bio-Rad Laboratories, Inc, CA). Protein samples (20 µg each) were mixed with sample loading buffer, heated for 5 min at 95 °C and resolved by 12% SDS-PAGE. Cellular proteins were transferred to Immuno-Blot PVDF membrane (Bio-Rad Laboratories, USA) by electroblotting. The membranes were blocked with 5% non-fat milk in PBS for 1 h, followed by incubation with primary antibodies against p-SAPK/JNK, p-p38, p-NF-κB, SAPK/JNK, p38 and NF-κB in 1:1000 dilution at 4 °C overnight. All primary antibodies were purchased from Cell Signaling (Cell Signaling Technology, Inc). The blots were then washed four times with TBS and incubated for 2 h with HRP-conjugated secondary antibody (Promega, Madison, WI, USA). Immunoreactive bands were developed using an Amersham ECL plus Western Blotting Detection System (GE Health Care, Buckinghamshire, UK) and visualized by Molecular Imager ChemiDoc Imaging Systems (Bio-Rad Laboratories, Hercules, CA, USA)22 (link)–24 (link).
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