Enhanced chemiluminescent ecl reagent kit

The total protein in transfected KYSE450 and KYSE510 cells was extracted and quantified with the RIPA buffer (Solarbio, China), which was supplemented with 1% proteinase inhibitor cocktail. The protein concentration was obtained with the Pierce BCA protein assay kit (Thermo Fisher Scientific, USA). Next, the quantified protein was loaded into 10% or 12% SDS-PAGE for electrophoresis separation. The separated protein bands were then electronically transferred to the membranes (Sigma-Aldrich, USA). Following that, the membranes were put under ice for 2 h with 5% BSA at room temperature. This step was followed by the incubation of the membranes overnight with primary antibodies at 4 °C and 1.5 h incubation with secondary detection antibody (Cat# ab205718, Abcam, USA) at room temperature. Finally, the protein immunoblots were visualized using the Enhanced Chemiluminescent (ECL) Reagent Kit (Thermo Fisher Scientific. USA). The density of the blots was obtained using ImageJ software. All the primary antibodies were purchased from Abcam (USA), including anti-CyclinB1 (Cat# ab32053), anti-ICAM1 (Cat# ab53013), anti-VCAM1 (Cat# ab134047), anti-Cleaved PARP (Cat# ab32561), anti-Bax (Cat# ab32503), anti-Cleaved Caspase-3 (Cat# ab2302), anti-TXNRD1 (Cat# ab124954) and anti-β-actin (Cat# ab8227).

Roswell Park Memorial Institute medium (RPMI 1640 medium), fetal bovine serum (FBS), trypsin/EDTA reagents, 100× antibiotic–antimycotic, and insulin (human recombinant, zinc solution, 4 mg/mL) were purchased from Invitrogen Corporation (Carlsbad, CA, USA). Acrylamide and protein molecular weight standard markers were obtained from Bio-Rad (Hercules, CA, USA). Radioimmunoprecipitation assay buffer (10×; RIPA lysis buffer) was purchased from Millipore Corporation (Billerica, MA, USA). The Enhanced Chemiluminescent (ECL) reagent kit was purchased from Thermo Scientific Inc. (Rockford, IL, USA). Anti-cyclin-B1 and cyclin D1/2 antibodies were obtained from Millipore Corporation (Billerica, MA, USA). Anti-β-actin antibody (AC-15) was purchased from Sigma Aldrich (St. Louis, MO, USA).