Rabbit anti laminb1

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.

Protein extraction and quantification were performed following the manufacturer’s instructions for the reagents used. Each sample containing 40 µg of protein was resolved by 12% SDS-PAGE, and the samples were then electrophoretically transferred onto Immuno-Blot polyvinylidene fluoride membranes (Hybond Inc., Escondido, CA, USA). The membranes were blocked with 5% non-fat dry milk in TBS solution for 1 h at room temperature, followed by incubation overnight with primary antibodies, including rabbit anti-HMGB1 (1:8,000; Abcam), mouse anti-β-actin (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-Lamin B1 (1:500; Proteintech Group, Inc., Chicago, IL, USA) antibodies. The membrane was then incubated with a horseradish peroxidase-conjugated anti-IgG (1:2,000; CWBIO) for 1 h at room temperature. Subsequently, immunoreactive bands were visualized using an ECL kit (Beyotime Institute Biotech, Shanghai, China) and quantified by densitometry using the ECL-Plus detection system (ClinX Sciences Instruments, Shanghai, China). β-actin was used as controls for protein loading.