Proteinase inhibitor pmsf

HCT-8 and HCT-116 cells were lysed using the high RIPA lysis buffer with proteinase inhibitor PMSF (Beijing Solarbio Science & Technology Co., Ltd.) at a ratio of 100:1 (v/v), after transfection for 48 h. The protein concentration was determined using the bicinchoninic acid reagent kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 30 µg of protein was loaded per lane and separated using a 10% SDS-PAGE gel and subsequently transferred onto PVDF membranes (EMD Millipore). The membranes were blocked using 5% skimmed milk for 1 h at room temperature, followed by overnight incubation with primary antibodies against Snail (1:1,000; cat. no. 3879; Cell Signaling Technology, Inc.), E-cadherin (1:1,000; cat no. 3195; Cell Signaling Technology, Inc.) and β-actin (1:1,000; cat no. 3195; Cell Signaling Technology, Inc. x) at 4°C. The membranes were subsequently incubated with anti-rabbit horseradish peroxidase-labeled secondary antibodies (1:5,000; cat. no. SA00001-2; Wuhan Sanying Biotechnology) for 1 h at room temperature. Finally, the PVDF membranes were developed using the enhanced chemiluminescence method using Luminata™ Western HRP Substrates (EMD Millipore) and detected using a chemiluminometer (Tanon Science and Technology Co., Ltd.).

Human GC SGC-7901 and MGC-803 cell lines were obtained from the Chinese Academy of Sciences (Shanghai, China). EGb761 was purchased from Dr. Willmar Schwabe GmbH&Co/Co. (Karlsruhe, Baden-Württemberg, Germany). U0126 was from SelleckChem (Radnor, PA, USA). RPMI-1640 medium, fetal bovine serum (FBS), and 0.25% EDTA trypsin were all bought from Gibco (New York, NY, USA). Six-well plates, 24-well plates, and Matrigel were acquired from Corning (New York, NY, USA). Rabbit antibodies against ERK and p-ERK were obtained from Cell Signaling Technology (Boston, MA, USA), rabbit antibodies against MMP2 were gained from Abcam (Cambridge, UK), rabbit antibodies against NF-κB p-P65 were from Bioss (Beijing, China), and rabbit antibodies against NF-κB P65 and GAPDH were from Proteintech Group (Wuhan, China). The secondary antibody, horseradish peroxidase (HRP)-linked goat anti-rabbit IgG, was purchased from EarthOX (San Francisco, CA, USA). RIPA buffer and phosphate-buffered saline (PBS) were bought from LEAGENE (Beijing, China). Proteinase inhibitor (PMSF) and TBST were from Solarbio Biotech (Beijing, China). Phosphatase inhibitor was from Beyotime (Shanghai, China) and OB glue was from Baiyunshan (Guangzhou, China). The polyvinylidene fluoride (PVDF) membranes were purchased from Millipore (Billerica, MA, USA).