Hrp conjugated goat anti mouse antibody

Four human HCC and one normal liver cell lines were used in this study. SMMC-7721 and L02 cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Huh7 cell was from Riken Cell Bank (Tsukuba Science City, Japan). MHCC-97H and MHCC-97L cells were from Liver Cancer Institute, Zhongshan Hospital of Fudan University (Shanghai, China) [11] (link). SMMC-7721 and L02 cells were cultured in RPMI medium 1640 (GIBCO, USA). Huh7, MHCC-97H and MHCC-97L cells were cultured in DMEM supplemented with 10% fetal bovine serum (GIBCO, Austria). Cells were cultured in 37°C incubator with humidified atmosphere containing 5% CO₂. Goat anti-human Nova1 polyclonal antibody was purchased from LifeSpan Biosciences (Seattle, USA) for detecting Nova1 in HCC tissues and from Abcam (Cambridge, USA) for detecting Nova1 in cell lines. HRP-conjugated rabbit anti-goat antibody was purchased from EarthOx, LLC (San Francisco, USA). Mouse anti-GAPDH antibody, HRP-conjugated goat anti-mouse antibody and HRP-conjugated goat anti-rabbit antibody were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The enhanced chemiluminescence Western blotting Substrate System was purchased from Pierce (Rockford, USA).

To detect antigen expression following mRNA-LNP vaccination, 293T cells cultured in 24-well plates were incubated with the mRNA-LNP vaccine (1 µg per well) for 24 h. The cells were washed with cold PBS and lysed with RIPA lysis buffer (Beyotime). Equal amounts of protein were subjected to SDS-PAGE and electrotransferred onto nitrocellulose membranes (Pall, USA). The membranes were blocked in 5% nonfat milk for 2 h at room temperature and then incubated with an anti-PEDV-S monoclonal antibody (made in our laboratory). After three washes with PBST, the membranes were incubated with an HRP-conjugated goat anti-mouse antibody (Beyotime) for 1 h at room temperature. After washing, proteins were detected using an ECL kit (Tanon, China).

Autophagic activity of the cells was evaluated by LC3 (microtubule-associated protein 1 light chain 3) detection with immunoblotting after treatment with 50 nmol/L rapamycin (Sigma, CA, USA) for 2 h. Total cell proteins were extracted with RIPA buffer (Beyotime, Suzhou, China). Protein concentration was determined by BCA assay (Beyotime). Protein exacts were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime) and transferred onto ployvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Then, the membrane was blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 30 min at room temperature. After incubation with rabbit anti-rat LC3 antibody (1:500, Novus, Littleton, CO, USA) and mouse anti-rat β-actin antibody (1:3000, EMD Millipore) at 4 °C overnight respectively, the membrane was incubated with HRP-conjugated goat anti-rabbit antibody (1:5000) and HRP-conjugated goat anti-mouse antibody (1:1000; Beyotime) at room temperature for 2 h. After washing in TBST for three times, the blots was detected by BeyoECL (Beyotime) and imaged in ChemiDoc XRS+ system (Bio-Rad Laboratories, Hercules, CA, USA). Relative expression of LC3-II was shown as a ratio of LC3-II/β-actin.