Cytoplasmic and nuclear proteins were extracted from cells using a Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Protein concentrations were determined using a BCA assay. Western blotting was performed as previously described with minor modifications (18). Proteins (50 µg) were separated via 10% SDS-PAGE and transferred to PVDF membranes (20 V; 100 mA) overnight. Following blocking with 5% skimmed milk at 37°C for 4 h, the membranes were incubated with primary antibodies (all 1:1,000) targeted against: CD40 (cat. no. sc-59047), ICAM-1 (cat. no. sc-8439), VCAM-1 (cat. no. sc-13160), iNOS (cat. no. sc-7271), NF-κB p65 (cat. no. sc-166748), p-IκB-α (cat. no. sc-8404), IκB-α (cat. no. sc-1643) and IKK-β (cat. no. ab32135) at 4°C for 24 h. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies (cat. no. sc-2005; 1:800) at 25°C for 12 h. Protein bands were visualized using an ECL Plus kit and a Gel Imaging System (Thermo Fisher Scientific, Inc.). Protein expression was semi-quantified using Quantity One software (version 4.0; Bio-Rad Laboratories, Inc.). Histone and β-actin were used as the loading controls for nuclear and cytoplasmic proteins, respectively.

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