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Fig. 5. Activity of the anaphase-promoting complex or cyclosome (APC/C) in response to DNA damage with and without mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B)
via an in vitro ubiquitination assay. HeLa cells were either transfected with the control small interfering RNA (siRNA) oligonucleotide (10 nM) or Mad2B siRNA oligonucleotide (10 nM). Then, 24 h post-transfection, the cells were treated with 50 μM cisplatin for varying time periods (0–12 h). APC/C was immunopurified from the control and Mad2B-depleted cells. As a positive control, APC/C was immunopurified from mitotic HeLa cells. Immunopurified APC/C was then normalized by quantification of the protein concentration. A ubiquitination mixture containing E1, E2, ubiquitin, ATP, Myc-tagged Cyclin B N-terminal residues 1-90 (Myc-CycB N90) as a substrate, and 1.5 μM of Cdc20 protein were added. Ubiquitinated Cyclin B (CycB N90) was analyzed via Western blotting with an anti-Myc tag antibody and the quantitated data of ubiquitinated CycB is shown in the lower panel. Lane 1, APC/C without Cdc20 addition (negative control); Lanes 2, 3, and 4, transfection with the control siRNA oligonucleotide; Lanes 5, 6, and 7, transfection with Mad2B siRNA oligonucleotide; Lane 8, APC/C from Nocodazole (0.2 μg/ml for 16 h)-arrested HeLa cells.
Korean J Physiol Pharmacol 2023;27:427-436
© Korean J Physiol Pharmacol