Anti e6

For whole cell lysates, cells were resuspended in 1X SDS sample buffer (25mM Tris–HCl pH 6.8, 1.5mM EDTA, 20% glycerol, 2% SDS, 5% b-mercaptoethanol, 0.0025% Bromophenol blue). For Western blot analysis, equal quantity of cell lysates were separated by SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane (VWR) and probed with the following primary antibodies: anti-NF-YA (Santa Cruz, sc-17753), anti-NF-YB (GeneSpin), anti-p53 (Santa Cruz, sc-126), anti-PARP1 (Santa Cruz, sc-8007), anti-H2AX (Santa Cruz, sc-101696), anti-p21 (Millipore, 05-345), anti-E2F1 (Bethyl, A300-766A), anti-cJun (Bethyl, A302-958A), anti-cMyc (Santa Cruz, sc-764), anti-Fos (Santa Cruz, sc-52), anti-p63 4A4 (Santa Cruz, sc-A0311), anti-actin (Santa Cruz, sc-1616), anti-tubulin (Sigma Aldrich, T-6074), anti-E6 (Santa Cruz, sc-365089). Chemiluminescent detection reagent was purchased from Millipore Spa (Luminata Classico and Forte Western HRP).

Total protein from cells were extracted by using RIPA buffer (50 mM TrisHCl, 150 mM NaCl, 2 mM EDTA, 1% NP-40 and 0.1% SDS). Total protein concentration was measured by using the BCA protein assay (Beyotime, Shanghai, China). Samples containing 30 μg protein was loaded in each lane, separated on 10% SDS PAGE gel and then transferred onto nitrocellulose membranes for a conventional western blot analysis. Antibodies used include Anti-E6 (1:500, sc-460, Santa Cruz Biotechnology, Santa Cruz, CA, USA) anti-DNMT1 (1:2000, ab87656, Abcam, Cambridge, UK), anti-E-cadherin (1:1000, #3195, Cell Signaling, Danvers, MA, USA) and anti-N-cadherin (1:1000, #13116, Cell Signaling). β-actin served as loading control and was detected by using ant-β-actin (1:500, ab8229, Abcam). Membranes were washed and incubated with corresponding HRP-labeled secondary antibodies. Protein signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, lL, USA) and intensity of each band was quantified by ImageJ software. Results were shown by three independent experiments (n = 3).