Ecl assay kit

Total cellular protein was extracted and separated by 10% SDS-PAGE then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% non-fat dry milk in PBST for 1 h at room temperature and then incubated with the primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 2 h at room temperature. Specific protein bands were visualized using the enhanced chemiluminescent (ECL) assay kit (Thermo Scientific, PA, United States). The following primary antibodies were used for western blotting, E-cadherin (3195), N-cadherin (13116), Vimentin (5741), β-catenin (8457). All antibodies were purchased from Cell Signaling.

Total protein was extracted from three infected CRC cell lines (HCT116, RKO, and MC38) using RIPA lysis buffer supplemented with 1% PMSF. Total protein was separated using a 10% sodium dodecyl sulfate‒polyacrylamide gel and then transferred to a PVDF membrane. The membranes were blocked with 5% skim milk for 2 h and then incubated with rabbit anti-C6orf15 (Proteintech, USA; 1:1000 dilution), rabbit anti-β-catenin (Proteintech, USA; 1:1000 dilution), rabbit anti-ZEB1 (Abmart, Shanghai; 1:1000 dilution), rabbit anti-E-cadherin (Abmart, Shanghai; 1:1000 dilution), rabbit anti-N-cadherin (Abmart, Shanghai; 1:1000 dilution), rabbit anti-Vimentin (Proteintech, USA; 1:1000 dilution), rabbit anti-ZO-1 (Abmart, Shanghai; 1:1000 dilution), mouse anti-GAPDH (Abmart, Shanghai; 1:3000 dilution), rabbit anti-CPT1A (Proteintech, USA; 1:1000 dilution), and rabbit anti-LaminB1 (Proteintech, USA; 1:1000 dilution). The membranes were then incubated with secondary antibodies for 2 h. We assessed the protein blotting results using an enhanced chemiluminescence (ECL) assay kit (Thermo Fisher Scientific, Rockford, IL, USA), and each experiment was performed three times.

L-MSCs were homogenized in lysis buffered (Tris-HCl 50 mM, pH 7.4, TBS) supplemented with Mini Tablet Protease Inhibitor Cocktail (Sigma-Aldrich) and whole cell lysate protein concentration was determined using a Nanodrop. 30μg of total protein was subjected to SDS-PAGE. The separated proteins were transferred electrophoretically to PVDF membranes (Millipore, Bedford, MA) using a semi-dry transfer blot system and blocked in TBS containing 5% non-fat dried milk powder for 1h and then incubated with the SOX-2 primary antibody overnight. The blots were then incubated with secondary antibody conjugated to horseradish peroxidase in the same buffer for 1h. Peroxidase-labeled proteins were visualized using an enhanced chemiluminescence (ECL) assay kit (Thermo Fisher). Blots were re-probed for β-actin (Sigma-Aldrich) to control for equal loading of samples. The relative intensities of the bands were quantified by densitometry using the NIH Image J software.