Western blot ecl substrate kit

The L1:P18I10 VLPs purified by ultracentrifugal and chromatographic methods were mixed with 2× Laemmli sample buffer (BIO-RAD, Hercules, CA, USA) in the presence or absence of 20 mM dithiothreitol (DTT) and reacted at room temperature (RT) for 15 min. Samples were separated by 8–16% TGX stain-free protein gels (BIO-RAD). Then, the gels were transfer to PVDF membranes. The membranes were probed with the anti-HPV16 L1 CAMVIR-1 mAb at a dilution of 1:4000. After that, the membranes were incubated with anti-mouse IgG Peroxidase Conjugate (Sigma-Aldrich, St. Louis, MO, USA) at a dilution of 1:4000. The signal was developed and visualized by chemoluminiscence using Western Blot ECL substrate kit (Bio-Rad). The blot images were acquired by using Odyssey Fc imaging system.

Trizol reagent, BCA Protein assay kit, 0.05% trypsin-EDTA, and RIPA lysis buffer were purchased from Thermo Fisher Scientific (Waltham, MA). Protease inhibitor cocktail was purchased from Roche Diagnostics (Mannheim, Germany). Lipofectamine™ 3000 transfection reagent was bought from Invitrogen (Carlsbad, USA). Polyvinylidene fluoride (PVDF) membrane, western blot ECL substrate kit, and blotting-grade blocker were purchased from Bio-Rad (Hercules, CA, USA). In vivo-jetPEI^TM was purchased from Polyplus-transfection (NY, USA). Alanine aminotransferase (ALT) assay kit (C009-2-1), aspartate aminotransferase (AST) assay kit (C010-2-1), blood urea nitrogen (BUN) assay kit (C013-2-1), creatinine (Cr) assay kit (sarcosine oxidase) (C011-2-1), total bilirubin (TB) assay kit (C019-1-1) and cardiac troponin-I (cTnI) assay kit (H149-1) were all purchased from Nanjing Jiancheng Bioengineeing Institute, Nanjing, China. All other solvents and chemicals of analytical grade were purchased from either Thermo Fisher Scientific or Sigma-Aldrich (St. Louis, MO).

The HPV16 L1, L1:P18I10 and L1:T20 VLPs were mixed with 2× Laemmli sample buffer (BIO-RAD) in the absence or presence of 5% (v/v) 2-mercaptoethanol (2-ME) and reacted at room temperature (RT) for 24 h. Samples were separated by 8–16% TGX stain-free protein gels (BIO-RAD). Then, the gels were transferred to PVDF membranes. The membranes were probed with the anti-HPV16 L1 CAMVIR-1 mAb at a dilution of 1:4000. After that, the membranes were incubated with anti-mouse IgG Peroxidase Conjugate (Sigma-Aldrich, St. Louis, MO, USA) at a dilution of 1:4000. The signal was developed and visualized by chemoluminiscence using Western Blot ECL substrate kit (Bio-Rad, Hercules, CA, USA). The blot images were acquired by using Odyssey Fc imaging system.