Nb100 40845

1×10⁷ cells were treated by 400 μL cell lysis buffer containing 1x protease and phosphatase inhibitor cocktail for 30 min on ice with the gently and repeatedly pipette-blowing, then centrifuged with 14000 rpm for 15 min at 4° C. The total protein supernatant was determined the concentration. 40 μg total protein for each sample was added 1x protein loading buffer, and processed under metal bath at 100° C for 10 min, followed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were transferred to polyvinylidene fluoride membranes. The membranes were then blocked by 5% non-fat milk TBS for 90 min and then incubated with the respective primary antibodies of DTO1L (1:2000, Cat. No. NB100-40845, Novus) and β-Actin (1:5000, Cat. No. AF5001, Beyotime Biotechnology) overnight at 4° C. Membranes were washed in TBST and incubated with rabbit anti-mouse and goat anti-rabbit IgG-HRP at room temperature for 1 h. After that membranes were incubated with 3 ml of ECL Western Blotting Substrate for 5 min in darkness at room temperature and developed the bands.