Goat anti rabbit immunoglobulin g

The human colon cancer cell line Caco2 was obtained from the American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin mixture, trypsin-EDTA, and fetal bovine serum (FBS) were obtained from Gibco Life Technologies (Milano, Italy). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). The protein assay kit was bought from Bio-Rad (Berkeley, CA, USA). An enhanced chemiluminescence kit, and rabbit monoclonal antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and EGFR were purchased from Proteintech (Chicago, IL, USA) and Abcam (Cambridge, UK), respectively. Goat anti-rabbit immunoglobulin G was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Sigma-Aldrich, Gillingham, UK) and EGFR Human Gene Knockout Kit [clustered regularly interspaced short palindromic repeats (CRISPR)] were purchased from Sigma-Aldrich and OriGene (Rockville, MD, USA), respectively. The Caco2 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37% and 5% CO₂ in humidified atmosphere. The culture medium was changed every other day, and the cells were subcultured using trypsin-EDTA. We obtained 5-FU from Sigma-Aldrich Co.