Anti phospho nf κb p65 ser536 93h1

For cytoplasmic and nuclear extracts, Caco‐2 cells were harvested, washed in cold PBS twice, and centrifuged at 3,300 g for 5 min in cold room to collect pellets. Pellets were resuspended in double cell volume of cytoplasmic extract (CE) buffer [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.3% NP‐40, and 1× protease inhibitor cocktail (Thermo Scientific™)], incubated on ice for 10 min and centrifuged at 800 g for 5 min to obtain the supernatants as cytoplasmic fraction. The pellets (containing the nuclei) were resuspended in equal volume of nuclear extract (NE) buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 25% glycerol, and 1× protease inhibitor cocktail), incubated on ice for 10 min, and centrifuged at 16,100 g for 5 min to obtain the supernatants as nuclear fraction. The protein concentrations were measured by Bradford protein assay (Protein Reagent, Bio‐Rad). For Western blots, cytoplasmic and nuclear proteins were fractionated on 10% SDS–PAGE and transferred to nitrocellulose blotting membranes (Amersham™Protran™ 0.45 μm NC, GE Healthcare, Life science). Membranes were incubated with anti‐phospho‐NF‐κB p65 (Ser536; (93H1), Cell Signaling, #3033), 1:1,000, Lamin B1‐Nuclear Envelope Marker (Abcam, ab16048) 1:5,000, and anti‐β‐actin (Cell Signaling, #4970) 1:1,000.