Ram hrp

U2OS cells were harvested in lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl pH 8.0; Sigma-Aldrich) supplemented with 1x PIC-C (Calbiochem), incubated on ice for an hour, then centrifuged (13,000 rpm at 4 °C for 5 minutes). The supernatant lysates were mixed with the same amount of 2x SDS loading buffer containing 5% β-mercaptoethanol (Sigma-Aldrich) and boiled for 5 minutes. The lysates were separated in SDS-PAGE, transferred to Amersham Hybond ECL-membrane (GE Healthcare) and incubated with the following primary antibodies: anti-p53 (Dako, IS616), anti-S15 P p53 (Cell signalling, 9284), 1BP7G5 anti-RPB1 (from L. Tora, IGBMC), anti-S2P RPB1 (Abcam, ab5095) and anti-S5P RPB1 (Abcam, ab5131), anti-GAPDH (Millipore, MAB374); then the following secondary antibodies: RAM-HRP (Dako, P0260) and GAR-HRP (Dako, P0448). Chemiluminescent detection was conducted using Immobilon Western Chemiluminescent HRP substrate (Millipore).

Cell were harvested in lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl, pH 8.0, and 1× PIC; Sigma-Aldrich, St. Louis, MO, USA) and incubated on ice for 1 h, followed by centrifugation (6000 rpm at 4 °C for 10 min). The supernatant lysate was supplemented with 6× SDS loading buffer containing 5% β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and boiled for 10 min. The samples were separated by SDS-PAGE and transferred to Amersham Hybond ECL-nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). The following first antibodies were used: anti-H3 (Abcam, ab1791, Cambridge, UK) in 1:3000 dilution and anti-GFP (Abcam, ab6556, Cambridge, UK) in 1:1000 dilution. For chemiluminescent detection, secondary antibodies were applied: RAM-HRP (Dako, P0260, Santa Clara, CA, USA) and GAR-HRP (Dako, P0448, Santa Clara, CA, USA), followed by incubation with Immobilon Western Chemiluminescent HRP substrate (Merck Millipore, Burlington, MA, USA) and scanning using the Li-Cor 3600 C-DiGit Blot Scanner platform (Li-Cor, Lincoln, NE, USA).