Odyssey infrared imaging system

Cells were washed twice with cold PBS (pH 7.4) and RIPA lysis buffer mixed with phosphatase inhibitor, and phenylmethanesulfonyl fluoride was then added to the cell samples, followed by centrifugation at 14,000 g for 15 min at 4°C to extract total proteins. Protein concentrations were measured using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Jiangsu, China), and protein samples were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk/Tris-buffered saline–Tween for 2 h, and then incubated with the following primary antibodies: PKC, phospho-IκB, phospho-ERK, phospho-JNK, phospho-p38, IκB, ERK, JNK, p38, and GAPDH (1:500–1000 dilution) overnight at 4°C. The membranes were subsequently washed three times with Tris-buffered saline–Tween and incubated with secondary IRDye^®-conjugated goat anti-mouse or goat anti-rabbit antibodies (LI-COR Biosciences, Lincoln, NE, United States) at 1:8000 dilution for 50 min at room temperature. The membranes were washed a further three times, and the bands were scanned and their intensities measured using an Odyssey infrared imaging system (Gene Company, Beijing, China).

After lysing cells with extraction buffer, cell extracts were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. The following primary antibodies were used: rabbit anti-eNOS, KDR, Cyr61, Myl9, MMP9, ANGPTL3, and VEGF (abcam); EPHB4, RXRα, ERα, pAKT, AKT, pY925 FAK, FAK, pERK, ERK, and mouse anti-GAPDH (Santa Cruz Biotechnology). Antibody incubations were performed overnight at 4 °C. The secondary antibodies used were IRDyeTM-800-conjugated anti-mouse and anti-rabbit IgG (Li-COR Biosciences). Immunoreactivity was detected using an Odyssey Infrared Imaging System (Gene Company Limited). All immunoblots were repeated at least two–three times.

Colon samples were homogenized and lysed in SDS-PAGE sample buffer, boiled, centrifuged and supernatant recovered. Samples were run on 10% SDS polyacrylamide gels, electroblotted onto nitrocellulose membranes, incubated with blocking buffer for 1h. Immunoblotting was assayed using anti-SCF (1:200) antibodies. Anti-β-actin (1:200) was used as loading control. The detection and analysis were done by Odyssey Infrared Imaging System (Gene Company Ltd., Hongkong, China).

The protein in the cell lysates was separated via SDS-PAGE and transferred to nitrocellulose membranes (micropores). Primary antibodies (Abcam) targeting the following proteins were applied: IKKβ (cat. no. ab124957, 1:2000), cyclinD1 (cat. no. ab16663, 1:2000), Bcl-2 (cat. no. ab59348, 1:2000), IL-8 (cat. no. ab18672, 1:2000), and GAPDH (cat. no. ab9485, 1:1000). An IRDye-labeled donkey anti-mouse or rabbit anti-IgG (Licor Biosciences) was used as the secondary antibody, and the membrane was assayed with an Odyssey Infrared Imaging System (Gene Company Limited).

Protein samples were extracted from fresh hippocampal tissue using the Total Protein Extraction Kit (KGP2100, KeyGEN BioTECH, China). Protein concentrations were determined using the BCA Protein Assay Kit (KGPBCA, KeyGEN BioTECH, China). Samples were then electrophoresed by SDS-PAGE and transferred to 0.2 μm PVDF membranes (10600021, Amersham, Germany), which was then cut according to protein marker. Membranes were blocked in 5% bovine serum albumin (BSA) at room temperature for 2 h and incubated in the relevant primary antibodies at 4°C overnight. After washes (3 times 10minutes) in 0.1% tris-buffered saline supplemented with Tween-20 (TBS-T), membranes were incubated in appropriate secondary antibodies at room temperature for 2 h. The protein bands were visualized in an Odyssey Infrared Imaging System (CLX-0796, Gene Company Limited, USA) and quantified with ImageJ software (National Institutes of Health). We used the following antibodies: anti-PSD95 antibody (1 : 2000, ab18258, Abcam, UK), Total OXPHOS Rodent WB Antibody Cocktail (1 : 1000, ab110413, Abcam, UK), Beta Actin Mouse Monoclonal antibody (1 : 5000, 66009-1-Ig, Proteintech, USA), goat anti-mouse (1 : 5000, 926-32210, LI-COR, USA), and goat anti-rabbit (1 : 5000, 926-32211, LI-COR, USA).

After each group LFBs were treated at a certain concentration for 48 h, followed by a mixture of the BRIPA lysis buffer and a protease inhibitor cocktail (GenDEPOT; P3100, P3200) to prepare the whole cell proteins.
We measured the cell protein concentrations with a bicinchoninic acid assay kit. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. We incubated the membrane with the following antigen recognition antibody at 4^oC overnight: α-SMA, fibronectin, collagen I, E-cadherin and XBP-1,blocking with 5% nonfat milk later. Incubation of membrane with polyclonal anti rabbit/mouse or goat HRP binding secondary antibody (1:5000 dilution; LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature was done, after washing with Tris-buffered saline Tween. Next, we used Tris-buffered saline-Tween to wash the membranes 3 times, and using an ECL detection solution to detect. We used the Odyssey infrared imaging system (Gene Company, Beijing, China) to scan and quantify the band intensities.