Proteinase inhibitor cocktail

SW480 cells were lysed in lysis buffer containing Proteinase Inhibitor Cocktail (KeyGEN BioTECH, Nanjing, China) and Halt Phosphatase Inhibitor Cocktail (KeyGEN BioTECH). Protein concentrations were quantified with a BCA protein kit (KeyGEN BioTECH). Samples (30 μg protein per lane) were loaded into 8–10% SDS–PAGE gels. After electrophoresis, the proteins were transferred from the gel to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk and incubated at 4 °C overnight with primary antibodies [β-actin polyclonal antibody (Bioworld, Irving, TX, USA; 1:5000), Claudin-2 antibody (Affinity, Cincinnati, OH, USA;1:500), Hes-1 polyclonal antibody (Abclonal, Wuhan, China; 1:500), Notch-1 polyclonal antibody (Abclonal, 1:500), and ZO-1 polyclonal antibody (Abclonal, 1:200)]. Immunoreactivity was detected by incubation with horseradish peroxidase-conjugated secondary antibodies (BOSTER, 1:2000) followed by chemiluminescent substrate development (Affinity). The results were detected by using a ChemiDoc XRS + with Image Lab Software (Bio-Rad). All samples were evaluated in parallel with three replicates.

Cells were cultured in 10 cm dishes for 48 h after transfection and were harvested using an IP lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40, 0.1% SDS) containing a proteinase inhibitor cocktail (Keygen). Protein lysates were incubated on a rotator with 1 µg of primary antibodies for 2 h at room temperature, followed by the addition of 30 µL of IP beads (Bimake, China) and incubation on a rotator at 4 °C overnight. The beads and immune complexes were subjected to washing steps with IP lysis buffer 5 times, with each wash involving rotation at 20 s per round for 5 min at room temperature. The samples were boiled in SDS loading buffer at 95 °C for 5 min. The immunoprecipitated samples were detected via western blotting.

Cells were lysed with RIPA buffer (Beyotime, China) containing a proteinase inhibitor cocktail (Keygen, China) and phosphatase (Beyotime). Protein sample concentrations were estimated using the BCA assay kit (Bio-Rad, USA). Protein samples were separated using a 10% SDS-PAGE gel and the separated proteins were subsequently transferred to a PVDF membrane (Millipore, USA). The membrane was blocked using 5% milk for 1 h at room temperature and was probed with primary antibodies at 4 °C overnight. Next, the membranes were incubated with the peroxidase-conjugated secondary antibody for 1 h at room temperature. GAPDH was used as the protein loading control. The blots were developed using the eECL Western Blot Kit (CWBIO Technology, China) and imaged using a chemiluminescence imaging system MiniChemi™ 610 with SageCapture^TM software (SAGECREATION, China). The antibodies used are listed in Supplementary Table 4.