For western blotting, cell lysates and protein samples were subjected to SDS-PAGE and transferred to PVDF membrane (Bio-Rad, USA). Membranes were blocked in PBS-T containing 5% w/w non-fat milk (BD DifcoTM, USA). The rabbit polyclonal antibodies raised against HPV16 E7 were used as the primary antibodies. The peroxidase-conjugated anti-rabbit immunoglobulin G (IgG; diluted 1:5000 in PBS; Beyotime) was used as the secondary antibodies. Protein was detected with Enhanced Chemiluminescence (ECL) western blotting substrate (Millipore, USA) through a chemiluminescence imaging system (Fujifilm, USA).
For immunofluorescence, 293T cells and SiHa cells were fixed in 4% paraformaldehyde (Bio-Rad, USA) and permeabilized with 0.1% Triton X-100 (Solarbio, China). Anti-HPV16 E7 polyclonal antibody was used as the primary antibody and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:500; Beyotime) was used as the secondary antibody. DAPI (Beyotime) was applied to stain the nucleus and stainings were observed under an immunofluorescence microscope (Olympus, USA).
For immunofluorescence, 293T cells and SiHa cells were fixed in 4% paraformaldehyde (Bio-Rad, USA) and permeabilized with 0.1% Triton X-100 (Solarbio, China). Anti-HPV16 E7 polyclonal antibody was used as the primary antibody and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:500; Beyotime) was used as the secondary antibody. DAPI (Beyotime) was applied to stain the nucleus and stainings were observed under an immunofluorescence microscope (Olympus, USA).