Fig. 2. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) binds to cell division cycle (Cdc)-20 following cisplatin addition.
(A) HeLa cells were transfected with HA-Mad2B for 48 h. Cdc20 was immunoprecipitated from the transfected cells following cisplatin treatment (50 μM for 12 h). Immunoprecipitates were immunoblotted with antibodies against HA (middle panel), Cdc20 (upper panel), and γ-tubulin (lower panel). (B) HA-Mad2B co-localizes with Cdc20 following cisplatin addition. HeLa cells were co-transfected with Myc-Cdc20 and HA-Mad2B for 24 h and then treated with cisplatin (50 μM) for 24 h. Cells were stained for HA-Mad2B (green), DNA (blue), and Myc-Cdc20 (red). Scale bar, 10 μm. (C) Glutathione S-transferase (GST)-pull-down assay. Cell lysates were prepared from exponentially growing cells (interphase) treated with cisplatin (50 μM) for 18 h. Purified GST-Mad2B proteins (0.2–1 μM) were added to the cell lysates. Samples were incubated before the addition of glutathione-sepharose beads. Proteins bound to the beads were immunoblotted with the anti-Cdc20 antibody. As a negative control, lysis buffer was used for the pull-down assay (buffer only). As a positive control, total cell lysates (5% input) were used for identifying Cdc20 on the Western blots (WB). This result is representative of three independent experiments. IP, immunoprecipitation; HA, hemagglutinin.
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