Phenylmethanesulfonylfluoride fluoride

Cells were harvested and lysed with Western/IP lysis buffer (Beyotime, Haimen, China) containing the protease inhibitor phenylmethanesulfonylfluoride fluoride (Beyotime). The protein concentration was determined using the Enhanced BCA Protein Assay Kit (Beyotime). Proteins were denatured at 100 °C for 10 min, subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to 0.22 mm polyvinylidene fluoride membranes (Merck-Millipore, Darmstadt, Germany). The membranes were blocked using 5% non-fat milk and 1% Tween 20 in phosphate-buffered saline (PBS) for 1 h and incubated with primary antibodies at 4 °C overnight. The membranes were subsequently washed and incubated with the appropriate secondary antibodies at room temperature. Finally, the signals were detected by standard analysis of HRPO-induced chemiluminescence. Details of the antibodies used in western blot are listed in Supplementary Table 1.

The cell lysates were prepared using RIPA cleavage buffer and phenylmethanesulfonylfluoride fluoride (Beyotime Biotechnology, China), to extract the proteins from the cells after full cleavage. The protein was fractionated by SDS-polyacrylamide gel electrophoresis, electroblotted on 0.45 μm polyvinylidene difluoride membranes (Millipore, USA), blocked with 5 % skimmed milk powder at 37 °C for 1 h, washed three times with 1 × PBS-T (1000 mL 1×PBS + 0.5 mL Tween-20), and incubated with the primary antibody (diluted 1:1000) overnight at 4 °C. After three washes, a secondary antibody (diluted 1:10,000) was added to the membrane and incubated for 2 h, and the membrane was washed five times. Finally, an ECL kit (Beyotime Biotechnology, China) was used for detection. Anti-STIL and anti-GAPDH antibodies were purchased from Proteintech. A goat anti-mouse IgG (H + L) -HRP secondary antibody was purchased from Jackson ImmunoResearch. Each experiment was repeated three times.

Total proteins were extracted using an SDS Lysis Buffer (Beyotime Biotechnology) supplemented with 1% phenylmethanesulfonylfluoride fluoride (Beyotime Biotechnology) and 1% phosphatase inhibitor (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). To separate nuclear and cytoplasmic proteins, the Minute™ SC-003 kit (Invent Biotechnologies, Inc., Beijing, China) was used according to the manufacturer’s instruction. Proteins were run on 10% SDS-PAGE and transferred into a PVDF membrane, followed by incubation with a primary antibody overnight. The following primary antibodies were used: rabbit anti-CROT (1:1000 dilution), rabbit anti-LaminB1 (1:2000 dilution), rabbit anti-Bcl-2 (1:2000 dilution), and mouse anti-GAPDH (1:5000 dilution) from Proteintech Group, Inc (Wuhan, China) and rabbit anti-Bax (1:2000 dilution), rabbit anti-Smad4 (1:2000 dilution), mouse anti-Smad2 (1:2000 dilution), rabbit anti-phospho-Smad2 (1:2000 dilution), and mouse anti-β-actin (1:5000 dilution) from Cell Signaling Technology, Inc. (Danvers, MA, USA). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit IgG and anti-mouse IgG (1:10,000 dilution, Proteintech). Signals were detected using BeyoECL Moon (Beyotime) and quantified using ImageJ software.