Cullin4a

For Western blot, cell lysates (30 mg) were loaded on SDS-PAGE gels and transferred onto nitrocellulose membranes (Millipore), which were then incubated with the indicated primary antibodies overnight. Corresponding secondary antibodies were incubated with the membranes for 1 h, and the membranes were then photographed with a Tanon 5200 visualizer (Shanghai, China). Primary antibodies to the following proteins were used: UBC12, UBA3, cullin1, cullin2, cullin5, p21, NOXA (Abcam), NAE, cullin3, NEDD8, cullin4A, p27, Wee1, p-H3, ORC1, CDT1, p-H2AX, t-H2AX, ATF4, CHOP, DR5, cleaved-PARP, cleaved-caspase 8, and cleaved-caspase 3 (Cell Signaling, Boston, MA); cullin4B (Protein Tech); and β-actin (Protein Tech). For CHX-chase assays, cells were treated with 50 μg/mL CHX (Sigma) for the indicated times, and the band density in Western blot was quantified in Image J software.

Antibodies specific to C-PARP (Cell Signaling Technology, 5625), Cullin1 (Abcam, 75817), Cullin2 (Abcam, 166917), Cullin3 (Cell Signaling Technology, 2759), Cullin4A (Cell Signaling Technology, 2699), Cullin4B (Proteintech, 12916-1-AP), Cullin5 (Abcam, 184177), NAE (Cell Signaling Technology, 14321), NOXA (Cell Signaling Technology, 14766), P27 (Cell Signaling Technology, 3686), RBX1 (Abcam, 133565), UBA3 (Abcam, 124726), UBE2F (Abcam, 12932), and UBE2M (Abcam, 109507) were purchased commercially.