Nuclear and cytoplasmic protein extraction and Western blot

For distinguishing phosphorylation of AKT in the nuclear and cytoplasmic fraction, RPE-1 cells were collected and extracted by using a nuclear and cytoplasmic protein extraction kit (Beyotime; P0027), according to the manufacturer’s instructions. Briefly, cells were collected by PBS that contained 10 mM EDTA and washed one time by PBS. Cells were lysed in ice-cold cytoplasmic protein extraction fraction A for 15 min and centrifuged at 15,000 g for 5 min; the supernatant fraction was cytoplasmic protein. The pellet was then resuspended in the ice-cold nuclear protein extraction buffer by vortexing, incubated on ice for 30 min and centrifuged at 15,000 g for 10 min; the supernatant fraction was the nuclear protein. To quantify protein concentration, a BCA kit for protein determination (ZOMANBIO; ZD301-2) was used. For the Western blot assays, the proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% milk in PBST (PBS with Tween 20), probed with primary and then secondary antibodies, and finally exposed using ECL (Bio-Rad; US EVERBRIGHT). The concentration of primary antibodies AKT1, β-tubulin, β-actin, pericentrin, and 53BP1 was 1:3,000, and the concentration of another primary antibody was 1:1,000. The secondary antibody HRP-Rb/M was purchased from Jackson ImmunoResearch and the concentration was 1:20,000.