Hrp linked anti mouse igg

The primary antibodies used in the western blot analyses include anti-BRD4N (targeting BRD4 156–284, 1:40,000), anti-BRD4C (targeting BRD4 1313–1362, 1:40,000), anti-Cyclin B1 (1:500, sc-245, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GAPDH (1:4000, G8140-01, US Biological, Salem, MA, USA), anti-Actin (MAB1501, Millipore, Burlington, MA, USA) and anti-HA-HRP (1:2000, 12013819001, Roche, Rotkreuz, Switzerland). HRP-linked anti-rabbit IgG (1:3000; 7074S; Cell Signaling Technology, Danvers, MA, USA) and HRP-linked anti-mouse IgG (1:3000; 7076S; Cell Signaling Technology) were used as secondary antibodies. Western blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA), and images were captured using an Amersham Imager 600 (GE Healthcare). Detailed information about western blot can be found in Figures S6 and S7.

Western blotting was performed as described previously [62 (link)]. The primary antibodies used in this study include anti-γH2A.X (1:1000, 2577S, Cell Signal Technology, Danvers, MA, USA), anti-p53 (1:500, sc-6243, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p53ser15 (1:1000, 9286S, Cell Signal Technology, Danvers, MA, USA), anti-cleaved PARP1 (1:1000, 5625S, Cell Signal Technology, Danvers, MA, USA), anti-MCL1 (1:1000, 39224S, Cell Signal Technology, Danvers, MA, USA), anti-BCL2 (1:1000, 2872S, Cell Signal Technology, Danvers, MA, USA), anti-ACTIN (1:150,000, MAB1501, Millipore, Burlington, MA, USA). The secondary antibodies used were HRP-linked anti-rabbit IgG (1:4000, 7074S, Cell Signaling Technology, Danvers, MA, USA) and HRP-linked anti-mouse IgG (1:4000, 7076S, Cell Signaling Technology, Danvers, MA, USA).

Whole cell extracts and immunoblotting were performed as described earlier (8 ). Soluble proteins were prepared by centrifugation of cell lysates for 15 min in a microfuge at 4 °C, 18,000g. To assay insoluble proteins, remaining cell pellets were resuspended in solubilization buffer (20 mM phosphate buffer pH 8.0, 300 mM NaCl, 2% SDS, 2 mM DTT, 1% Triton X-100, 8 M urea, and 1× protease inhibitor cocktail) and incubated at room temperature for 5 min, followed by centrifugation at 18,000g for 15 min at 4 °C in a microfuge. The supernatant was then heated with in sample buffer (Bio-Rad, Cat. No. 1610737) for 5 min at 37 °C. Antibodies recognizing the following epitopes were used for immunoblotting at the indicated dilutions: Acss2, 1:1000 (Cell Signaling Technology, Cat. No. 3658); GFP, 1:1000 (Invitrogen, Cat. No. A11121); α-tubulin, 1:5000 (Sigma, Cat. No. T9026); V5, 1:1000 (Invitrogen, Cat. No. R96025); LC3B, 1:1000 (Cell Signaling Technology, Cat. No. 2775); ubiquitin, 1:1000 (Cell Signaling Technology, Cat. No. 3936); HRP-linked anti-mouse IgG (Cell Signaling Technology, Cat. No. 7076); HRP-linked anti-rabbit IgG (Cell Signaling Technology, Cat. No. 7074).