Odyssey infrared imaging system clx 0796

The HUVECs were treated with SDG or DMSO (1%) for 1 h and then treated with LPS or PBS for 6 h. Total or nuclear proteins of colonic tissue or cells were prepared and extracted using the Protein Extraction Kit (KeyGEN Biotechnology Co., Ltd., Jiangsu, China). Then, the concentration of protein was measured using the BCA Protein Assay Kit (KGP902). 50 μg protein was resolved by a 10% sodium dodecyl sulfate- (SDS-) polyacrylamide gel (SDS-PAGE) to evaluate the expression of the protein and then transferred onto 0.22 μm polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After blocking with 5% nonfat milk, the membrane incubated with primary antibodies overnight at 4°C as follows: rabbit anti-NF-κB p65 (1 : 1000), p-NF-κB p65 (1 : 1000), p-IκB-α (1 : 1000), IκB-α (1 : 1000), p-Akt (1 : 1000), β-actin (1 : 3000), and PCNA (1 : 1000). After washing for 3 × 5 min in PBST (contain 1‰ tween20), goat anti-rabbit IgG (H&L) secondary antibody conjugated (1 : 5000) was incubated for 1 h at RT. Then, results were detected using an Odyssey Infrared Imaging System CLX-0796 (LI-COR, Lincoln, NE, USA), and semiquantitative analysis was then performed using Image J software.

Total protein from the hippocampal tissue and BV2 microglial cells was prepared and extracted using the BCA Protein Extraction Kit (KGP2100, KeyGEN Biotechnology Co., Ltd, Jiangsu, China). Protein concentration was measured using the BCA Protein Assay Kit (KGP902, KeyGEN Biotechnology Co., Ltd, Jiangsu, China). Equal amounts of protein (50 µg per lane) were resolved on a 10% or 12% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE), and then transferred onto 0.22-μm polyvinylidene fluoride (PVDF) membranes (Millipore, USA) blocked with 5% non-fat milk, and incubated with the following primary antibodies overnight at 4°C: rabbit anti-Bcl-2 (1:500), Bax (1:1000), cleaved caspase-3 (1:1,000), NLRP3 antibody (1:500), caspase-1 p20 (1:500), ASC (1:500), IL-18 (1:500), IL-1β (1:500), TNF-α (1:1,000), and β-actin (1:2,000). Membranes were washed with TBST (TBS containing 1‰ Tween 20) and subsequently incubated with Goat Anti-Rabbit IgG (H&L) Secondary antibody Conjugated (1:5,000). Detection was performed with an Odyssey Infrared Imaging System CLX-0796 (LI-COR, Lincoln, NE, USA) and quantified via densitometry with Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). All experiments were performed in triplicate.