Western blot analysis of NF-κB p65 and NF-κB phosphorylated (p)p65

The BxPC-3 cells were seeded at 2×10⁶ in 100-mm dishes with 10% FBS and cultured to ~80% confluence. The cells were then treated with escin (10 µM) in 5% FBS for 2 h and stimulated with or without TNF-α (5 ng/ml) for 30 min before the end of the incubation. Nuclear and cytoplasmic extracts were obtained from the cells using a Nuclear Extraction kit (Active Motif, Inc.). The concentrations of each protein were measured with a Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc.). A total of 20 µg each protein extract was denatured at 90°C for 5 min and separated on 10% Mini-PROTEAN TGX Precast gels (Bio-Rad Laboratories, Inc.). The protein bands were transferred to nitrocellulose membranes and blocked in iBind Flex Solution (iBind Flex Buffer, iBind Flex Additive and distilled water; Thermo Fisher Scientific, Inc.) for 15 min at room temperature. The primary and secondary antibody reactions were performed using the iBind Flex Western System (Thermo Fisher Scientific, Inc.) for 2.5 h at room temperature according to the manufacturer's instructions. The membranes were incubated with anti-p65 (1:1,000; cat. no. 8242S; Cell Signaling Technology, Inc.), -p-p65 (1:1,000; cat. no. 3033S; Cell Signaling Technology, Inc.), -GAPDH (1:2,000; cat. no. SC-47724; Santa Cruz Biotechnology Inc.) and -TATA-box binding protein (TBP; 1:1,000; cat. no. 22006-1-AP; ProteinTech Group, Inc.) primary antibodies, then HRP-conjugated goat anti-rabbit polyclonal secondary antibody (1:2,000; cat. no. P0448; Agilent Technologies, Inc.). The protein-antibody complexes were visualized with a SuperSignal West Pico Chemiluminescent Substrate, SuperSignal West Femto Chemiluminescent Substrate, or Pierce ECL Western Blotting Substrate (all from Thermo Fisher Scientific, Inc.). The immunoreactive protein bands were detected using an Amersham Imager 600 (Cytiva).