Depletion of topoisomerase II leads to shortening of the metaphase interkinetochore distance and abnormal persistence of PICH-coated anaphase threads

JCS013730 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Western blot showing robust knockdown of endogenous topo IIβ 72 hours following transient transfection of siRNA, both in the presence and absence of doxycycline (72 hours) to deplete topo IIα. To examine siRNA knockdown of topo IIβ, proteins were resolved using an 8% SDS-PAGE gel, transferred to nitrocellulose using a buffer containing methanol and SDS, and stained/fixed in Ponseau S/acetic acid. The following antibodies were used: human topo IIβ (1:1000, BD Biosciences) and α-tubulin (1:50,000) (Abcam). Detection, using anti-mouse horseradish peroxidase (1:2000), was by the ECL Plus Western Blotting Analysis System (Amersham BioScience).