Cell Lysis and Protein Extraction

The details of sample treatment were described previously (18). Treated and untreated cells were washed three times by ice-cold PBS buffer to remove the cell culture medium. The denaturing buffer containing 8 M urea and 1 M ammonium bicarbonate was added to each cell culture dish for cell lysis. Cell lysate of each sample was sonicated by ultrasonic cell distribution system at 60% power for 10 min (10 s break after each 8 s sonication) in an ice bath until the solution became clear. The samples were then centrifuged at 14,000g for 15 min at 4°C and the supernatants were collected. The protein concentration was measured by BCA protein assays (Beyotime, Shanghai, China). Cellular proteins were harvested from three biological replicates at each condition, and the proteins were pooled into one sample for further sample preparation.