Hpv16e6

Whole cell lysates were mixed with Laemmli loading buffer, boiled, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to a PVDF membrane. Subsequently, immunoblot analyses were performed using antibodies specific to p53 (Calbiochem, San Diego, CA), HPV16E6 (Abcam, Cambridge, MA), HPV16E7 (Cervimax, Vienna, Austria), or GADPH (Cell Signaling Technology, Danvers, MA). The signal was developed with ECL (Thermo Fisher Scientific) after incubation with appropriate secondary antibodies.

Cells were lysed in RIPA lysis buffer (Pioneer Technology, Xi'an, China) with protease inhibitor cocktail tablets and phosphatase inhibitor cocktail tables (Roche). Then cell lysates were centrifuged at 12,000 r.p.m (revolutions per minute) for 40 min at 4°C. The supernatant was harvested and the protein concentration was determined using a Pierce BCA protein assay kit (Thermo scientific, Fremont, USA). An equivalent amount of protein was separated by SDS-PAGE and transferred to polyvinyldifluoride membranes (Millipore, Billerica, USA). The membranes were probed with the primary antibodies overnight at 4°C, followed by a secondary anti-rabbit or mouse IgG conjugated with HRP. Signals were detected using chemiluminescence reagent (Millipore) and ChemiDoc System (Bio-Rad, Hercules, USA). Primary antibodies were used at a 1:1000 dilution and the antibodies were against: HPV16 E6 (ab70, Abcam, USA), p75NTR (MAB367, R&D, USA), PI3K (#4249, CST, USA), Akt (#9272, CST, USA), p-Akt(ser473) (#4060, CST, USA), GAPDH (#5174, CST, USA), β-actin (#3700, CST, USA) was used as the endogenous reference. Secondary antibodies used were horseradish peroxidase (HRP) - conjugated anti-rabbit (#4414, CST, USA) and anti-mouse (#4410, CST, USA).