Cullin1

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.

Cell lysates were prepared for immunoblotting analysis using antibodies against LC3, p62, NEDD8, AMPK, p-AMPKα (Thr172), ULK1, p-ULK1 (Ser317), p-H2AX, WEE1, p21, ORC1, Beclin1, ATG5, p-p70S6K (Thr389), p70S6K, 4EBP1, p-4EBP1 (Thr37/46), cleaved PARP, cleaved Caspase-3, IκBα, p-IκBα, p65, LaminA/C and Tublin (Cell Signaling Technology), Cullin1 (Abcam). ACTIN (Protein Tech) was used as the loading control.

MLN4924 was purchased from Med Chem Express. Antibodies (Abs) for immunoblotting were all obtained from Cell Signaling Technology, except Deptor (Proteintech Group), Cullin-1 (Abcam, Cambridge, MA), cyclin-D1 and cyclin-D3 (Beyotime Institute of Biotechnology). The HRP-linked secondary antibodies were purchased from Sigma-Aldrich. Antibodies for APC-CD11c, PE-CD80, PE-CD86 and FITC-CD4 were purchased from eBioscience. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was obtained from Invitrogen.