Chemiluminescence imaging system

Cell lysates or protein samples prepared in reducing sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 0.01% Bromophenol Blue, 10% glycerol, 50 mM DTT) were subjected to SDS-PAGE and transferred to polyvinylidene fluoride (0.2 μm PVDF, Bio-Rad, U.S.A.) membranes. After blocking with 10% nonfat milk (BD Difco^TM, U.S.A.) for 1 hour, the membranes were incubated with antibodies specific for HPV58-E7 (1:10,000). Secondary peroxidase-conjugated goat anti-rabbit IgG (1:5000; Beyotime) was incubated for 2 hours at room temperature and ECL substrates (Millipore, U.S.A.) were applied and signals were detected by a chemiluminescence imaging system (FUJIFILM, U.S.A.).

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 Difco^TM, 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).

Total cellular lysate and Rac1-GTPase pull-down complex were separately loaded for running a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). Followed by the completion of electrophoresis, the gel was transferred onto a polyvinylidene difluoride (PVDF) membrane by using semi-dry transferring method (Bio-Rad). The transferred membrane was blocked with 5% non-fat milk (in phosphate buffered saline with Tween-20 (PBST) buffer) for 1 hour and then washed three times for 5 minutes with PBST prior to adding the diluted primary antibody (1:1000 in PBST). The hybridization was undergone for one hour and the hybridized membrane was washed with PBST three times for 5 minutes prior to adding the diluted secondary antibody (1:5000 in PBST). At last, the chemiluminescent reagent was added to the washed membrane and the image was developed by the Chemiluminescence Imaging System (Fuji, Japan) as manual’s instruction.