Anti bubr1

HeLa cells were cultured in DMEM containing 10% FBS in a humidified atmosphere containing 5% CO₂. After treatment with each compound for 2.5 h (interphase MT network) or 5 h (mitotic spindle), the cells were immediately fixed with cold MeOH (−20 °C). After blocking with PBS containing 0.5% bovine serum albumin, cells were incubated with anti-α-tubulin antibodies (1:500 dilution, Santa Cruz, Cat# sc-32293) to observe interphase cells, or with anti-BubR1 (1:250 dilution, BD Bioscience, Cat# 612502) and anti-α-tubulin (1:250 dilution, MBL, Cat# PM054) antibodies to observe mitotic spindle. After staining with Alexa⁴⁸⁸-conjugated anti-mouse IgG (1:2,000 dilution, Invitrogen, Cat#A11001) and Alexa568-conjugated anti-rabbit IgG (1:2000 dilution, Invitrogen, Cat#A11011), cells were washed four times with PBS and mounted with PBS containing 0.1 μg ml⁻¹ DAPI. MTs and BubR1 signals were observed under a Leica LAS AF 6000 fluorescence microscope (Leica Microsystems) or an LSM 700 laser-scanning confocal microscope (Zeiss).

Cells were plated in 6 wells at a density of 2 × 10⁵ cells per well. After PARP inhibition, cells were washed twice with PBS and resuspended in 200 μl of TR3 Lysis Buffer (3% SDS, 10% Glycerol, 10mM Na₂HPO₄ anhidro). Then cells were sonicated and 20 μl of 50% β-mercaptoethanol - 50% Bromofenol blue were added. The protein concentration was determined using the Lowry assay. Proteins were resolved on SDS-polyacrylamide gels and transferred onto PVDF Membrane (Biorad). The blot was blocked with 5% milk powder in PBS1X with 0.1% Tween-20 for 60 minutes and incubated overnight with 1% milk powder in PBS1X with 0.1% Tween-20 with the following antibodies: anti-PARP-1(C2-10 mouse, ALEXIS, LA), anti-PTEN (Santa Cruz Biotechnology sc-7974), anti-BRCA1 (Santa Cruz Biotechnology sc-642), anti-RAD51 (Santa Cruz Biotechnology (H-92) sc-8349), anti-phosphoERK (Santa Cruz Biotechnology sc-7383), anti-ERK (Invitrogen. Carlsbad, CA 61-7400), anti-phospho H2AX (Millipore 05-636) and anti-BUBR1 (BD Bioscience. Erembodegem, Belgium). α-Tubulin (Sigma, St Louis MO) and β-Actin (Sigma, St Louis MO) were used as loading control. Bands were visualized with ECL, ECL-PLUS and ECL PRIME (Amersham Biosciences) and the pictures were taken with the imaging system ChemiDoc XRS System (BIO-RAD) and medical X-ray films (AGFA).