Secondary goat anti mouse igg antibody

At 72 h after transfection, aliquots of 5 × 10⁶ cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology, China), and the lysate was determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). A total of 30 μg protein per lane was resolved by 10% SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, USA). The membrane was blocked with 5% non-fat milk and then incubated overnight with primary antibodies against β-actin (Sigma-Aldrich, St. Louis, Missouri, USA), CDK-4 (Cell Signaling Technology, USA), CCND1 (Cell Signaling Technology, USA), or CCND2 (Cell Signaling Technology, USA). The membrane was washed in TBST and incubated with the secondary goat anti-mouse IgG antibody (Beyotime Institute of Biotechnology, Shanghai, China). An enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) was used to detect the protein bands using a FluorChem E System (ProteinSimple, CA, USA).

ELISA was used to measure antigenicity of JEV-ΔNS1 and JEV-specific IgG antibody levels of vaccinated mice as described previously^{17 (link)}. The purified viral particles (WT JEV or JEV-ΔNS1) dissolved in lysis buffer (10% glycerin and 1% Triton X-100 in deionized water) were diluted with coating buffer (0.085 M sodium bicarbonate and 0.015 M sodium carbonate, pH9.5) to 100 ng/ml. One hundred microliters of diluted volumes (10 ng) were added to each well in a 96-well plate. The coated 96-well plates were then blocked with PBS containing 5% skimmed milk. The fourfold serial dilutions (starting at a 1:50 dilution) of heat-inactivated sera from vaccinated mice were added to the coated plates, followed by incubation with the HRP-conjugated goat anti-mouse IgG secondary antibody, and visualized using a two-component 3,3′,5,5′-tetramethylbenzidine (TMB) color development kit (Beyotime Biotechnology). After the addition of 1 M H₂SO₄ stop solution, the optical density at 450 nm was measured using a multimode microplate reader (Varioskan Flash; Thermo Fisher) according to the manufacturer’s instruction. The IgG antibody titers were defined as the highest dilution of sera giving an optical density twice that of the nonimmune sera.