Ecl detection reagent

Cells were harvested, washed and suspended in RIPA lysis buffer (Wuhan Servicebio Technology Co. Ltd.). Following 6000g centrifugation for 10 min at 4 °C, the supernatant was collected. The total protein in each sample was quantified using a bicinchoninic acid (BCA) kit (Wuhan Servicebio Technology Co. Ltd.). Equal amounts of proteins (10 µg/lane) were electrophoretically separated via 12% SDS-PAGE and transferred to a PVDF membrane (EMD Millipore). The membrane was blocked for 1 h in 5% skimmed milk dissolved in 0.1% of Tween-20 used for TBST at 4 °C and then incubated with antibodies directed against, human caspase 3 (1:1000), c-caspase 3 (1:1000), IκB (1:1000), p-IκB (1:1000), NF-κB p65 (1:1000), BCL2 (1:1000), BCL-XL (1:1000), BAX (1:1000), AIP (1:1000), p53 (1:1000), and β-actin (1:1000) at 4 °C overnight, followed by incubation with goat anti-rabbit IgG H&L (HRP) (1:1000; A0216, Beyotime Institute of Biotechnology) or goat anti-rat IgG H&L (HRP) (1:1,000; A0208, Beyotime Institute of Biotechnology) at room temperature for 1 h. The protein signals were detected using Super electrochemiluminescence (ECL) Detection Reagent (Yeasen Biotechnology Co. Ltd) by 5.2 Image Lab software (Bio-Rad Laboratories, Inc.). The bands on the membrane were quantified by normalization to β-actin.

The total protein was extracted from the colonic tissue using a lysis buffer. The protein concentration of the supernatant was determined using a BCA protein assay kit (Vazyme, USA). Samples (25 µg of protein) were separated on 10% SDS-PAGE gels and transferred to PVDF membranes and incubated with skim milk (5%) for 1 h at room temperature and then with primary antibodies overnight at 4°C. The primary antibodies included those that were selectively recognized: TGF-β, Col1a2, Col3a2, α-SMA (Abcam, Cambridge, MA, USA), followed by incubation with corresponding secondary antibodies. The signals were visualized using an ECL detection reagent (Yeasen Biotechnology, Shanghai, China), and the results were measured using ImageJ software 1.48 (National Institutes of Health).

Changes in the protein levels among different groups were verified by Western blot. For this purpose, total protein was extracted from aortic tissues from each diet group using a lysis buffer, and the protein concentration in the supernatant was calculated utilizing a BCA protein assay kit (Vazyme, USA). Then, 25 µg of protein from each group was separated using 10% SDS-PAGE gels, to be subsequently relocated to PVDF membranes. This was first incubated with skim milk (5%) for 1 h at room temperature, then with primary antibodies for Galectin-3 (1:5000, ab76245, Abcam) and Vcam1 (1:4000, ab134047, Abcam) at 4 °C, overnight, and finally with the correspondent secondary antibodies. Visualization was performed using an ECL detection reagent (Yeasen Biotechnology, Shanghai, China), and measurements were made by ImageJ software 1.48 (National Institutes of Health) [43 (link)].