Anti mouse horseradish peroxidase hrp linked secondary antibodies

HCT-116/HT-29 cells (2 × 10⁶ cells) were seeded in a 100-mm dish. After treatment with luteolin and/or selumetinib for 48 h, cells were collected in PBS and lysed with radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, Beijing, China) on ice for 30 min, and lysates were harvested by centrifugal separation. Protein concentration was determined using the BCA Protein Assay Kit (Takara). Identical amounts of protein were separated using 10% sodium lauryl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were clogged with 5% nonfat dry milk for 1 h at room temperature. The primary antibodies BCL-2, BAX, cleaved caspase-3, cyclin B1, p-CHK1, CDC2, XRCC1, LIG1, HMGB1, ERCC3, p-MEK1, MEK1, p-ERK1/2, ERK1/2, and GAPDH (1:1000 dilution, Sigma-Aldrich) were added after overnight incubation at 4°C. After three washes with TBST, the membranes were incubated with anti-mouse horseradish peroxidase (HRP)-linked secondary antibodies (Beyotime Institute of Biotechnology, Beijing, China) for 1 h. The intensity of protein expression was measured using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, USA) [31 (link),32 ].

The cells were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime) containing protease and phosphatase inhibitors on ice for 30 min. Protein concentration was determined using a BCA Protein Assay kit (Takara). Next, 50 μg of proteins was separated on 10% sodium lauryl sulphate-polyacrylamide gels (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). The membranes were blocked with 5% nonfat dry milk, and primary antibodies against ST3GAL3, cyclinD, cyclinE, PCNA, and GAPDH (1 : 1000, Abcam) were added overnight at 4°C. After three washes with Tris-HCl-buffered saline with 0.1% (v/v) Tween 20 (TBST), the membranes were incubated with anti-mouse horseradish peroxidase- (HRP-) linked secondary antibodies (Beyotime) for 1 h at room temperature. The intensity of protein expression was measured using an enhanced chemiluminescence reagent (Thermo Fisher Scientific).