After treatment, cells were collected and lysed in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1.0% NP-40, 0.1% SDS, and 0.5% deoxycholic acid) supplemented with protease and phosphatase inhibitors. Lysates were resolved in 10% SDS-PAGE gels for western blotting. Proteins were transferred to PVDF membrane; after blocking with 5% non-fat milk, membranes were incubated with primary antibodies: Cdc34 (Santa Cruz, sc-28381, 1:1000 dilution), p27 (BD Biosciences, 610242, 1:1000 dilution), p27 (Cell Signaling, 3686, 1:1000 dilution), SKP2 (Santa Cruz, sc-7164, 1:1000 dilution), SKP1 (BD Biosciences, 610530, 1:1000 dilution), and β-actin (Sigma, A5316, 1:50,000 dilution). Thereafter, membranes were washed and incubated with relative secondary antibodies: goat anti-rabbit secondary antibody (Cell Signaling, 7074, 1:5000 dilution), or goat anti-mouse secondary antibody (Cell Signaling, 7076, 1:5000 dilution).
Finally, the signals were visualized by the chemiluminescence system (Perkin Elmer). These experiments were conducted three times. Western blot band quantification was performed using Quantity One (BioRad Laboratories, Inc.) and signals were normalized to the control group. Unprocessed scans of blots are provided in the Source Data file.
Finally, the signals were visualized by the chemiluminescence system (Perkin Elmer). These experiments were conducted three times. Western blot band quantification was performed using Quantity One (BioRad Laboratories, Inc.) and signals were normalized to the control group. Unprocessed scans of blots are provided in the Source Data file.