Mouse anti hpv16 e6

Proteins were extracted from cells or tissues and added to the RIPA buffer. Cracking on ice for 30_60 minutes. The supernatant was collected by centrifugation at 4°C and 12000 rpm for 15 minutes. BCA protein assay kit (Boster Inc, China) was used for protein quantification. SDS-PAGE gel electrophoresis was performed. Then, the protein was transferred to the nitrocellulose membrane and 5% skimmed milk powder blocked the nonspecific antigen. The primary antibody was added and incubated at 4°C overnight. After washing the membrane, horseradish peroxidase-labeled IgG (secondary antibody) was added at 37 °C for 1h. Densitometric analysis was performed by Image Lab. In the assay, antibodies included rabbit anti-hnRNP E1 (Abcam, UK. ab74793, 1:1000), mouse anti-HPV16 E2 (Abcam, UK. ab17185, 1:1000), mouse anti-HPV16 E6 (Abcam, UK. Ab70, 1:1000), mouse anti-β-actin (Boster Inc, China, 1:300).

The transplanted TC-1 tumors were harvested, fixed in a 10% formaldehyde solution, embedded in paraffin, and cut into slices using standard procedures. After antigen retrieval (10 mM sodium citrate buffer, pH 6.0), endogenous peroxidases were blocked by 3% hydrogen peroxide in PBS for 10 min. Tissue sections were treated with primary antibodies for anti-cleaved caspase-3 (Abcam., Cambridge, UK) and mouse anti-HPV16 E6 (Abcam., Cambridge, UK) diluted at 1:500 concentrations in blocking buffer for 24 h at 4 °C.
Sections were then treated with horseradish peroxidase (HRP)-conjugated secondary antibodies at 1:100 dilutions and visualized using DAB plus chromogen substrate (Dako, Agilent, CA, USA) and hematoxylin counterstain. For quantification, five fields of view from at least four separate tissue sections were counted for each group (n = 03) using image J software. The number of positive brown-stained cells over the total number of cells was estimated and used to determine the percentage (%) of the staining area.