Anti topo 1

P8-D6 was synthesized as recently described [31 (link)]. The following antibodies, obtained from Santa Cruz (Heidelberg, Germany), were used for our experiments: anti-Topo I (sc-10783, rabbit polyclonal IgG, lot #D414), anti-Topo IIα (sc-5346, goat polyclonal IgG, lot #G2611), anti-Topo IIβ (sc-13059, rabbit polyclonal IgG, lot #D2214), anti-rabbit (sc-2357, mouse polyclonal IgG, HRP-coupled, lot #B0817), and anti-goat (sc-2354, mouse polyclonal IgG, HRP-conjugated, lot #H2213). ECL detection reagent and nitrocellulose membranes were ordered from GE Healthcare (Buckinghamshire, UK). Agarose was purchased from Bio-Rad (Vienna, Austria), and ethidium bromide and etoposide from Sigma-Aldrich (Taufkirchen, Germany). Live Cell Imaging Solution (LCIS) and all media and supplements for cell culture were obtained from GIBCO Invitrogen (Karlsruhe, Germany).

Cellular protein was harvested and protein contents were determined as previously described [17 (link)]. The same amounts of protein for each sample were loaded and separated by SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis). Separated proteins were transferred to a PVDF (Polyvinylidene fluoride) membrane (Hybond-P, Amersham GE Healthcare, Boston, MA, USA), blocked in TBST and 5 % milk powder, and incubated with primary antibodies (anti-Topo I 1:500 (Santa Cruz Biotechnology, #sc-271285, (Heidelberg, Germany)), anti-Topo II α/β 1:10000 (Abcam#ab109524, (Cambridge, UK)) and anti-HSP 90 1:10000 (Santa Cruz Biotechnology, #sc-13119, (Heidelberg, Germany)). Incubation with HRP-labeled anti-mouse IgG 1:2000 (Santa Cruz Biotechnology, #sc-516102 (Heidelberg, Germany)) or HRP-labeled goat anti-rabbit IgG 1:3000 (Elabscience #E-AB-1003 (Houston, TX, USA) followed. After washing with TBST, membranes were developed using the ECL Plus Western Blotting Detection System (GE Healthcare). HSP 90 was used as loading control. Chemiluminescence was visualized using the ChemoStar Touch ECL & Fluorescence Imager (Intas Science Imaging Instruments, Göttingen, Germany).

Cells lysates in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and protease inhibitors cocktail) were centrifuged at 15,000 rpm for 30 minutes at 4°C, and 1 mg protein of cleared lysates were used for each immunoprecipitation. The lysates were incubated overnight at 4°C with primary antibodies and then 30 μl of protein A/G PLUS-agarose beads (Santa Cruz) were added and incubation continued. Beads were washed with cold lysis buffer and boiled in SDS-PAGE sample buffer before elctrophoresis. Primary antibodies used were anti-ZFP91 [5 (link)], anti-HIF-1α (BD Biosciences; 610959 and Novus Biologicals; NB100-105), anti-VEGF (Santa Cruz; sc-152), anti-EPO (Santa Cruz; sc-80995), anti-CD31 (Santa Cruz; sc-1506), anti-p65 (Santa Cruz; sc-109), anti-Topo-I (Santa Cruz; sc-5342), anti-α-tubulin (Sigma; T5168).