Spindle checkpoint proteins are reduced in AEtr cells. (A) Western blot analysis of BubR1 and securin in K562 vector or AEtr-expressing cells treated with 0.025% DMSO (−) or 1 μg/mL nocodazole (+) for 18 hours. Immunoblot for tubulin indicates relative protein loading. Anti-HA Western shows AEtr expression. The same lysates were also analyzed for cohesin subunit Rad21 and tubulin to show relative protein loading. (B) Western blot analysis of separase full length and the autocleaved form in control or 2 different AEtr pools treated with nocodazole. The same membrane was probed for cohesin, which indicates the cleaved cohesin form is detectable only after a long exposure compared with cohesin in panel A. (C) K562 vector or AEtr-expressing cells were transfected with plasmids encoding EGFP or securin-EGFP. At 48 hours after transfection, cells were treated with 1 μg/mL nocodazole for 18 hours. Cells were monitored for endogenous securin and transfected securin-EGFP expression by immunoblotting, which detected expression of full-length securin-EGFP and C-terminal degraded forms (indicated by asterisks) as well as endogenous securin. (D) Cells were stained with propidium iodide to measure DNA content by flow cytometry. The percentage of cells in mitosis following transfection of EGFP or securin-EGFP for 3 independent experiments is shown. Error bars show SD of the average from 3 independent experiments.