E2f1 c20

For immunoblots, cells were harvested in radioimmunoprecipitation assay buffer [50 mM tris-HCl (pH 8), 150 mM NaCl, 1% (w/v) Igepal CA-630, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.2 mM sodium orthovanadate, and protease inhibitor cocktails] and incubated on ice for 30 min before SDS–polyacrylamide gel electrophoresis. The following antibodies were used in immunoblots: p100/TSN (A302-883A, Bethyl Laboratories; RRID: AB_10631268), E2F1 (C20, Santa Cruz Biotechnology; RRID: AB_631394), E2F1 (A300-766A, Bethyl Laboratories; RRID: AB_2096774), HA (16B12, Covance; RRID: AB_10063630), FLAG (M2, Sigma-Aldrich; RRID: AB_262044), β-actin (AC-74, Sigma-Aldrich; RRID: AB_476697), H4R3me2s (ab5823, Abcam; RRID: AB_10562795), histone H4 (ab10158, Abcam; RRID: AB_296888), and SENP7 (donated by R. Hay, University of Dundee, UK).

ChIP assay was conducted following the manufacture’s protocol (Active Motif). Briefly, after arsenic treatment, cells (5 × 10⁷) were fixed with 1% formaldehyde at RT for 5 min, followed by nuclei extraction. ChIP assay was conducted overnight at 4 °C using 7 μg of DNA and 3 μg of control IgG or E2F1 (C-20, Santa Cruz) after enzyme digestion of chromatin at 37 °C for 30 min. We used protein G magnetic beads (Active Motif) to capture the ChIP product, which was digested with proteinase K after reversion of cross-links for 15 min at 95 °C. The isolated DNA was used as the template for PCR amplification. The primers used for Aurora-A promoter were: site 1 (−268 to −80), sense 5′-TGGTCCGGTTCTCTTGGTAT-3′ and antisense 5′-AAGCTTGACGCATTGGAGAT-3′; site 2 (−104 to +106), sense 5′-CGACGCGTTGGCTCCACCACTTCCGG-3′ and antisense 5′-CCAGGAGCTCAGCCGTTAGAATTCAAAGG-3′ (He, [22 (link)]).