Antibodies against p65

Curcumin (purity≥98%) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibody against TLR4 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against p65, occludin and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against zonula occluden-1(ZO-1) and MyD88 were purchased from Abcam (Cambridge, MA, USA). SYBR Green-based real-time PCR kit was purchased from Applied Biosystems (Foster City, CA, USA). TRIzol reagent and the cDNA synthesis kit were obtained from Invitrogen Life Technology (Carlsbad, CA, USA). ELISA kits for TNF-α, IL-1β and LPS quantification were purchased from R&D Systems (Minneapolis, MN, USA). TransAM NF-κB p65 ELISA kit was purchased from Active Motif (Carlsbad, CA, USA).

In brief, 40 μg protein was loaded to SDS-PAGE, and then, the PVDF membrane was used to transfer the protein. Membranes were blocked in 5% skim milk for 1 h. Antibodies against p65 (1 : 1000, Cell Signaling Technology, Beverly, USA), lamin B (1 : 1000, Cell Signaling Technology, Beverly, USA), and GAPDH (1 : 1000, Beyotime Biotechnology, Shanghai, China) were used to incubate the membranes for a whole night. Next day, the membrane was incubated with a HRP-conjugated secondary antibody for 1 h. The blots were visualized using the chemiluminescent detection kit (Pierce, Thermo Scientific, Waltham, USA).

The protein expressions of p65, p-p65, IκBα, Nrf2, and HO-1 were determined by Western blot analysis. Antibodies against p65, p-p65 and IκBα were purchased from Cell Signaling (Beverly, MA, USA), antibody against Nrf2 was purchased from Ruiying Biological, and antibodies against GAPDH, β-actin and β-tubulin were purchased from Bioworld. Tissues were lysed in RIPA buffer (Applygen Technologies) with phosphatase inhibitor tablet (Roche Diagnostics) and protease inhibitor (Cocktails, AMRESCO). Lysates were then centrifuged for 15 min at 12,000 × g at 4 °C and supernatants were collected and protein concentrations were determined by BCA Protein Assay Reagent (Pierce). Cell lysates (15-30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to nitrocellulose membranes (Millipore). For western blotting analysis, membranes were incubated with primary antibodies for overnight at 4ºC followed by incubation with a secondary antibody for 1h at r.t. Then the signals were detected by enhanced chemiluminescence according to the manufacturer’s recommendation.