Hpv18 e7

Equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto Hybond nitrocellulose membrane (Amersham biosciences), followed by immunoblot analysis. Antibodies used in this study were as follows: CREB1 (9197, CST), phospho‐CREB1 (9198, CST), HPV16 E6 (GTX132686, GeneTex), HPV16 E7 (sc‐65711, Santa Cruz), HPV18 E6 (sc‐365089, Santa Cruz), HPV18 E7 (ab100953, Abcam), Snail (3879, CST), Slug (9585, CST), GAPDH (G‐9, SCBT), GFP (sc‐9996, SCBT), and FLAG (F3165, Sigma). Western blots were visualized with species‐specific HRP conjugated secondary antibodies (Jackson ImmunoResearch) and ECL (Thermo/Pierce).

The protein concentrations were determined with a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). Then, equal amounts of the protein extracts (30 μg) were electrophoretically separated through a 10% sodium dodecyl sulfate polyacrylamide gel. The proteins were transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific, Waltham, MA, USA) for 2 h and blocked with 4% skim milk powder in Tris-buffered saline-Tween for another 1 h. The membranes were incubated with the following primary antibodies overnight at 4°C: GM-CSF antibody (1:1000; Affinity, Cambridge, UK), B7.1 antibody (1:1000; Affinity), HPV16 E7 antibody (1:300; Santa Cruz Biotechnology, Dallas, TX, USA), FLT3L antibody (1:1000; Abcam, Cambridge, MA, USA), HPV18 E7 (1:1000; Abcam), luciferase antibody (1:1000; Abcam) and β-actin antibody (1:1000; Wuhan Doctorate Bioengineering, Wuhan, China). After being washed with phosphate-buffered saline, the membranes were then incubated with secondary antibody (1:5000; Abcam) at room temperature for 1 h. Finally, the immunoreactivity was detected with the enhanced chemiluminescence reagent (Santa Cruz Biotechnology).