Bright ecl kit

Total protein was extracted from CRC cells using cell lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), and the BCA method was used to detect the protein. Total protein (30 µg/lane) was separated by 12% SDS-PAGE and the separated proteins were subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore). The PVDF membranes were blocked in blocking solution (Beyotime Institute of Biotechnology) for 60 min in a shaker at room temperature and incubated with primary antibodies against: Cyclin-dependent kinase 4 (CDK4; cat. no. ab137675), cyclin D (cat. no. ab62151), cyclin E (cat. no. ab33911), matrix metalloproteinase 9 (MMP9; cat. no. BA0573) and β-actin (cat. no. 4ab010745) overnight at 4°C on a shaker, all of which used primary antibody dilution buffer and were purchased from Beyotime Institute of Biotechnology. Following the primary incubation, membranes were incubated with HRP-conjugated affinipure rabbit anti-Goat IgG (H+L) (1:5,000; cat. no. SA00001-1; ProteinTech Group, Inc) at room temperature for 60 min. Protein bands were visualized using the bright ECL kit (Advansta, Inc. K-12045-D20, http://advansta.com/products/western-blot-substrate-WesternBright-ECL) and protein bands were detected using an Odyssey Infrared Imaging system (LI-COR Biosciences).