Hpv16 e6 e6 6f4

For Western blot analysis, cells were lysed using lysis buffer (10 mM tris-HCL pH 7.5, 50 mM KCl, 2 mM MgCl₂, 1% Triton X-100, 1 mM DTT, 1 mM PMSF and protease inhibitor cocktail (Roche)). Samples were incubated for 20 min on ice with intermittent mixing and were then centrifuged for 10 min. Protein samples were mixed with 4x Laemmli-buffer-containing β-mercaptoethanol and then heated at 95 °C for 5 min. The samples were separated by SDS-PAGE and subsequently transferred to a PVDF membrane by semi-dry transfer. Membranes were blocked and then incubated overnight with primary antibodies (HPV16 E6 E6-6F4 (Euromedex), HPV16 E7 NM2 (in-house produced), Ras (G12V mutant-specific) D2H12 (Cell signaling technologies), CDKN2A/p16^INK4a EPR20418 (abcam) or β-actin AC-74 (Sigma-Aldrich)). HRP-coupled secondary antibodies (IgG anti-Mouse IgG (H+L)-HRPO (Dianova), IgG anti-rabbit IgG (H&L)-HRPO (Rockland Immunochemicals)) were then incubated with the membrane for 1 h. Chemiluminescence signal was detected after addition of a chromogenic substrate (VWR International # RPN2232) and detected in a Biorad Chemiluminescence Detector (Biorad #ChemiDoc).

For protein expression analysis by Western blot, cells were lysed using lysis buffer consisting of 10 mM tris-HCL pH 7.5, 50 mM KCl, 2 mM MgCl₂, 1% Triton X-100, 1 mM DTT, 1 mM PMSF, and protease inhibitor cocktail (Roche, Basel, Switzerland). Samples were incubated for 20 min on ice with periodic mixing followed by 10 min centrifugation at 14,000× g at 4 °C. Proteins were mixed with 4× Laemmli-buffer-containing β-mercaptoethanol and then boiled at 95 °C for 5 min. Isolated proteins were separated by SDS-PAGE and then transferred to a PVDF membrane using semi-dry transfer. Membranes were blocked followed by overnight incubation at 4°C with primary antibodies (HPV16 E6 E6-6F4 (Euromedex, Souffelweyersheim, France), HPV16 E7 NM2 (in-house produced), p21 Waf1/Cip1 (F-5), P16INK4A (DCS-50)sc-65476, p53 (DO-1)sc-126 (Santa Cruz Biotechnology, Dallas, Texas), β-actin AC-74 (Sigma-Aldrich), GAPDH (GT239) (Biozol, Eching, Germany). HRP-coupled secondary antibodies (IgG anti-Mouse IgG (H+L)-HRPO (Dianova, Hamburg, Germany) were then incubated with the membrane for 1 h. After adding the chromogenic substrate ECL™ Prime Western Blotting detection reagents (Cytiva Europe GmbH, Freiburg im Breisgau, Germany), a chemiluminescence signal was observed in a Biorad Chemiluminescence Detector (BioRad ChemiDoc, Hercules, CA, USA).