Whole cell lysates

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 whole cell lysates (Beyotime, Shanghai, China) were used to harvest total protein whose protein concentrations were measured by the BCA™ Protein assay kit (Pierce, Appleton, WI, United States). Proteins were separated by SDS-PAGE using 10% acrylamide gels. After three washes in TBST, the membranes were incubated with appropriate dilutions of primary antibodies against Ki-67 (1:1000; Abcam, Cambridge, MA, United States), PCNA (1:1500; Abcam, Cambridge, MA, United States), and GAPDH (1:1500; Abcam, Cambridge, MA, United States).

Total protein was extracted from the cells using whole cell lysates (Beyotime, Jiangsu, China). The protein concentrations of individual samples were assessed using a standard bicinchoninic acid assay (Beyotime). For each sample, 60 μg of protein was separated on 10% SDS-PAGE gel (Bio-Rad), transferred onto a polyvinylidene difluoride membrane (catalogue number: 3010040001; Roche Applied Science, Mannheim, Germany)and blocked with 5% skimmed milk and 0.1% Tris-buffered saline-Tween 20 (TBST) at room temperature for 1.5 h. The membranes were washed in TBST three times and incubated overnight at 4°C with monoclonal rabbit anti-Notch1 antibody (1/1,000 dilution) and monoclonal rabbit anti-GAPDH antibody (1/8,000 dilution). The primary antibodies recognize human protein and were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The membranes were then washed with 1X TBST (Jiuzhou Tech, Beijing, China) and incubated with anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Inc.). The protein expression was evaluated using chemiluminescence and exposure to Kodak film.