Ecl chemiluminescence kit

The experimental proteins were extracted using the whole cell lysates (Beyotime, Shanghai, China) and their concentration were measured by BCATM Protein Assay Kit (Thermo Scientific, MA, USA). After dividing the proteins with SDS-PAGE, proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA), and blocked in 5% skim milk in 0.1% TBST at 4°C overnight. The proteins were probed with BMP1, VIM and GAPDH, after which they were incubated with secondary antibody. Proteins were visualized with an ECL chemiluminescence kit (Boster, Wuhan, China).

The heart sample was homogenized using metal beads in RIPA buffer with protease inhibitor (Beyotimem, Shanghai, China) and PMSF (Beyotime, Shanghai, China). The concentration of total protein was determined by the BCA (Boster Biological Technology, Wuhan, China) method. A 5× loading buffer (Beyotime, Shanghai, China) was added to the sample at the ratio of 4:1 and boiled for 5 min at 95 °C. Then, 30 μg protein of each sample was run on a 12% SDS-PAGE gel (Epizyme Biomedical Technology, Shanghai, China), transferred to PVDF membranes (Boster Biological Technology, Wuhan, China) and then blocked with 5% non-fat milk for 60 min. The membranes were incubated with primary antibodies against Gal-3 (ABclonal, Wuhan, China) or GAPDH (ABclonal, Wuhan, China) at 4 °C overnight, and then membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies (ABclonal, Wuhan, China) for 1 h. The blots were developed using the ECL chemiluminescence kit (Boster Biological Technology, Wuhan, China). The levels of target proteins were normalized to GAPDH.

Total protein was extracted from tissues of the rectum specimens by using whole cell lysates (Beyotime, China) and protein concentrations were assessed by using the pierce BCA Protein Assay Kit (Beyotime, China). For each sample, 40 μg protein was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel, transferred electrophoretically onto a polyvinylidene difluoride membrane, and blocked in 5% milk in 0.1% Tris-buffered saline-Tween 20 (TBST) at 4°C overnight. After 3 washes in TBST, membranes were incubated overnight at 4°C with the appropriate dilution of primary antibodies against NSE, S-100, and C-kit (AF645; R&D Systems, China Co., Ltd). The membranes were then washed with 1XTBST and incubated with secondary antibody (Cell Signaling Technology Inc, Danvers, MA). Signals were performed with ECL chemiluminescence kit (Boster, Wuhan, China) and exposed to X-ray films. Alfa-glyceraldehyde phosphate dehydrogenase (α-GAPDH) immunobloting was used as an internal control.