Pres2

Approximately 5×10³ cells per well were settled in a 24-well plate overnight and then transfected with 1 μg of plasmids per well. After incubation for 24 hours, the cells were fixed with 4% paraformaldehyde, treated with 0.5% Triton-X 100, blocked with 1% BSA at room temperature, and then incubated with specific primary antibodies against p65 (Santa Cruz, USA) or preS2 (Abcam, USA) at 4℃ overnight. Subsequently, the cells were incubated with an Alexa Fluor® 568 conjugated donkey anti-Rabbit IgG (H+L) secondary antibody or Alexa Fluor® 568 conjugate goat anti-Mouse IgG (H+L) secondary antibody (ThermoFisher Scientific, USA) for one hour and then incubated with DAPI for 5 minutes at room temperature. The expression of MHB and the subcellular localization of p65 were observed with a Zeiss confocal microscope (LSM 510 Meta, Germany). For the subcellular localization of MHBs, MHBs and ELP-1-CFP were cotransfected into SMMC-7721 cells and observed with a Nikon confocal microscope (A1R, Japan).

Halt protease and phosphatase inhibitor cocktail (Thermo Scientific, USA) were added to RIPA buffer (Millipore, Germany) at a ratio of 5:100; the mixture was used to lyse cells to prepare protein samples. Protein samples were separated in 10% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Germany). After blocking with 5% bovine serum albumin (BSA), the membranes were incubated with specific primary antibodies (1:1,000) and then HRP-conjugated secondary antibodies (1:5,000). The primary antibodies comprised antibodies against GAPDH (HuaAn Biotechnology, China), preS2 (Abcam, USA), p44/42, phospho-p44/42, p38, phospho-p38, IκB (Cell Signaling, USA) and BiP (Abcam, USA). The specific bands were detected with an EZ-ECL Kit (Biological Industries, Israel). The relative levels of target proteins to GAPDH were determined with ImageJ software.