Enhanced chemiluminescence

Colon samples were homogenized using a protein extraction kit, in which the phenylmethanesulfonyl fluoride, protease inhibitors, and phosphatase inhibitors were mixed (Solarbio Life Sciences, Beijing, China), and then centrifuged at 12,000g for 20 min at 4°C. The protein concentration was determined using a bicinchoninic acid protein determination kit (Yeasen Technologies, Shanghai, China). For Western blot analysis, 50 μg of protein extract was separated with 10% NuPAGE (NP0302BOX, Invitrogen) and then transferred to a polyvinylidene fluoride (PVDF) membrane. This was sealed with 5% skim milk for 1 h at 28°C, using anti-p65 (51-0500, Invitrogen), anti-IL-1β (MM425B, Invitrogen), anti-TNF-α (AMC3012, Invitrogen), anti-IκBα (MA5-16152, Invitrogen), anti-Cox-2 (PA5-17614, Invitrogen), and anti-β-actin (MA1-140, Invitrogen); incubated in the PVDF membrane; washed five times with 1× TBST; and then combined with Horseradish peroxidase (HRP) secondary antibodies (A32723, Invitrogen). Next, the mixture was incubated for 1 h at room temperature and the PVDF membrane was washed five times with 1× TBST. Antibody binding was observed using enhanced chemiluminescence (Solarbio Life Sciences, Beijing, China). Finally, ImageJ software (U.S. National Institutes of Health, Bethesda, MD, United States) was used to quantify protein expression.

The total protein was extracted from tissue homogenate or cell lysate via the RIPA buffer (Beyotime). After the centrifugation, the supernatant was harvested and quantified through BCA protein assay kit (Beyotime) according to the manufacturer’s instructions. Next, 20 μg of protein was separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked in the blocking buffer (Beyotime) for 1 h, and then incubated with primary antibodies for anti-CyclinD1 (ab226977, 1:300 dilution, Abcam, Cambridge, UK), anti-USP11 (ab109232, 1:3000 dilution, Abcam), or anti-β-actin (ab8227, 1:3000 dilution, Abcam) overnight, followed by incubation of horseradish peroxidase (HRP)-labeled IgG (ab6721, 1:10000 dilution, Abcam) for 2 h. β-actin was used as a loading control. The protein signaling was developed via Enhanced Chemiluminescence (Solarbio). Relative protein level was analyzed using QuantityOne v4.6 (Bio-Rad) and normalized to GAPDH.

Western blot analysis was performed using standard techniques. Briefly, cells (2 × 10⁶/well) were treated with or without SlyD (200 ng/mL) for 40 hours. Total protein was extracted using a lysis buffer (2% mercaptoethanol, 20% glycerol, and 4% SDS, in 100 mM Tris–HCl buffer, pH 6.8). Equal amounts of total protein (60 µg/lane) were separated and transferred to PVDF membranes (Bio-Rad, Hercules, CA). The membranes were incubated with primary antibodies overnight at 4 °C: rabbit monoclonal anti-CDX2 (1:2000, Abcam, USA), mouse monoclonal anti-VIL1 (1:2000, Origene, USA), rabbit monoclonal anti-TCTP (1:250, Abcam, USA) and then with the appropriate horseradish peroxidase-conjugated secondary antibody (Zhongshan Golden Bridge Biotechnology Co. Ltd, Beijing, China) and visualized by enhanced chemiluminescence (Solarbio, China).

Human brain vascular smooth muscle cells were lysed in RIPA buffer (Beyotime), and protein was obtained after a centrifugation at 10,000 × g for 5 min. The protein was quantified with a bicinchoninic acid kit (Thermo Fisher Scientific) according to the instructions. The samples (20 μg) were separated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred on nitrocellulose membrane (Bio-Rad Laboratories). The membranes were incubated in 3% bovine serum albumin (Solarbio) for 1 h, and then interacted with primary antibodies overnight and secondary antibody for 2 h. All antibodies were purchased from Abcam (Cambridge, United Kingdom), including proliferating cell nuclear antigen (PCNA) (ab152112, 1:2,000 dilution), Bcl-2 (ab194583, 1:500 dilution), Bcl-2-associated X (Bax) (ab53154, 1:500 dilution), cleaved poly-ADP ribose polymerase (PARP) (ab32064, 1:3,000 dilution), MCL1 (ab243136, 1:2,000 dilution), GAPDH (ab9485, 1:5,000 dilution), and horseradish peroxidase-labeled IgG (ab6721, 1:10,000 dilution). Next, the membranes were interacted with enhanced chemiluminescence (Solarbio), and the blots were analyzed via Quantity One software (Bio-Rad Laboratories) with GAPDH as a normalized reference.

Transfected ECA-109 and TE-1 cells were lysed using precooled RIPA lysis buffer (Beyotime, China), and the protein concentration was quantified with the BCA Protein Assay Kit (Beyotime, China). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 10%) was employed to separate the proteins, and then the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in a TBST solution for 1 h at room temperature and incubated with primary antibodies against Caspase 3 (1:500; ab13847, Abcam), Caspase 9 (1:2,000; ab32539, Abcam), Bax (1:1,000; ab32503, Abcam), Bcl-2 (1:2,000; ab182858, Abcam), E-cadherin (1:500; ab15148, Abcam), Vimentin (1:1,000; #12826, Cell Signaling Technology, Boston, USA), N-cadherin (1:1,000; #14215, Cell Signaling Technology, Boston, USA), and GAPDH (1:10,000; R1210-1, HuaBio, Hangzhou, China) at 4 °C overnight. Then, the membranes were incubated with an appropriate HRP-conjugated secondary antibody at room temperature for 2 h. Finally, the bands were visualized by enhanced chemiluminescence (Solarbio, China), and the intensity of the protein bands was analyzed using ImageJ software.