Htert

Total protein was extracted with RIPA buffer and boiled for degeneration. 30 μg of the protein were loaded into 7.5% SDS-PAGE. After being electrotransferred and blocking with 3% BSA, the membranes were incubated with primary antibodies. Rabbit against MUC1, CD63, E-cadherin, N-cadherin (1:1000; Proteintech, USA), hTERT (1:1000; Bioss, China), HSP70 (1:500; Boster, USA), TSG101 (1:1000; ABclonal, USA), β-actin (1:3000; Proteintech, USA) and mouse against CD9 (1:1000; Proteintech, USA) were used as primary antibodies. Anti-rabbit or anti-mouse IgG conjugated with HRP (1:5000; Abbkine, USA) was used as the secondary antibody. Enhanced chemiluminescence (Abbkine, USA) was used to display the bands.

Total protein from cultured cells was extracted in cell lysis buffer (PIERCE, Rockford, IL) and quantified using the Bradford method. Fifty micrograms of protein were loaded and separated on SDS–PAGE (12%). After transferring to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA), the membrane was incubated overnight at 4°C with antibody against E6 (1:100; Bioss Biotechnology Co., Ltd, Beijing, China), LKB1 (1:1000 Cell Signaling, USA), SP1 (1:100; Bioss), hTERT(1:200, Bioss), or mouse monoclonal antibody against beta-actin or Rabbit monoclonal antibody against GAPDH (1:500; Santa Cruz Biotechnology, Santa Cruz, CA). After incubation with peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology) at 37°C for 2 h, bound proteins were visualized using ECL (Pierce) and detected using BioImaging Systems (UVP Inc., Upland, CA). The relative protein levels were calculated by normalizing to beta-actin protein as a loading reference.