Ecl anti rabbit igg hrp

Samples were prepared similarly as previously described^{65 (link)} with slight modifications. In short, cells were washed once in PBS, lysed in RIPA, treated with benzonase (Sigma) and rotated at 4 °C after which they were stored at −20 °C. Protein concentration estimation was done using a BCA estimation kit (Pierce). Equal concentrations of protein were prepared in 4x Lammeli buffer and boiled for 10 min before loading on an SDS-PAGE gel. Resolved proteins were then transferred onto a nitrocellulose membrane (Amersham), blocked in 5% BSA/PBS and then incubated overnight with one of the following antibodies, washed in TBS-T and followed by the respective secondary Ab for 2 h. γ-tubulin was used last as a loading control: anti-Pdk4 (1:1000, Abcam, ab38242), anti-Serpinf1 (1:2000, Sigma-Aldrich, AV20020-50UG), anti-γ-tubulin (1:1000, Sigma-Aldrich, T6557), ECL anti-rabbit IgG-HRP (1:5000, GE Healthcare), ECL anti-sheep IgG-HRP (1:5000, GE Healthcare). Blots were developed with ECL substrate (Pierce) and the signal detected on a Fusion-FX7 Chemiluminescence detection system (PeQlab). Originals are provided in Supplementary Fig. S4. Each experiment involved biological triplicates, which were subsequently used for statistical testing.

Total protein was extracted from sub-confluent cells with 1% NP40 lysis buffer containing 150mM NaCl, 50mM TrisHCl, 10% glycerol, 1X Halt proteinase inhibitor cocktail, 5mM NaF, and 1mM NaOrthovanadate. For nuclear lysate preparation, nuclear complex Co-IP kit (Active Motif, #54001) was used. Protein concentrations were estimated using BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Antibodies cleaved-PARP (Cell Signaling, #9541), BRD4 (Cell Signaling, #13440), HP1α/CBX5 (Cell Signaling, #2616), H2B (Millipore, 07-371), H2BD (Thermo Scientific, PA5-30561), Phospho-CHK1 (Ser345) (Cell Signaling, #2348), CHK1 (Santa Cruz, sc-8408), and GAPDH (Millipore, MAB374), and secondary antibodies ECL anti-rabbit IgG HRP and ECL anti-mouse IgG HRP (GE Healthcare) were used at 1:5000 dilutions. The band was visualized using either Lumina Classico or Crescendo Western HRP substrate system (Millipore) depending on signal intensities. Experiments were repeated three times; shown are representative blots for each.