Anti cbp

HNPCs nuclear extracts were prepared as described previously [26 (link)]. HAT activity and HDAC activity assays were done using nuclear extracts following the protocols of the manufacturer (BioVision Biotechnology). For HAT activity assays, immunoprecipitations were done using anti-p300, anti-Tip60, anti-CBP, and anti-PCAF (Abcam, Cambridge, UK) with hNPCs nuclear extracts. Precleared nuclear extract was incubated with antibodies overnight with Protein G dynabeads (Thermo, Waltham, Massachusetts, USA) at 4°C. All samples were counted with a multipurpose scintillation counter, LS 6500 (Beckman, California, USA).

The obtained tissue samples were successively fixed, decalcified, and embedded in paraffin. The sections were deparaffinized and hydrated. For mouse tissue IHC assays, the sections were incubated in 10 mM citrate buffer and microwaved at 750 W for 30 min for antigen retrieval. The sections were treated with 3% H₂O₂ for 20 min and blocked with 5% normal goat serum for 1 h. Then, the sections were separately incubated with anti-PCAF (Santa Cruz, sc-13,124), anti-p300 (Abcam, ab275379), anti-CBP (Abcam, ab253202), anti-GCN5 (Santa Cruz, sc-365,321), anti-IL-6 (Abcam, ab-290,735), anti-IL-1β (Abcam, ab-283,818), or anti-TNF-α (Abcam, ab-1793) antibodies at 4 °C overnight. Secondary antibody incubation and color development were performed using the SP Rabbit & Mouse HRP DAB Kit (Cwbio, 2069 S) according to the kit protocol.
Immunohistochemistry (IHC) semiquantitative analysis was performed with ImageJ software. Briefly, color separation was first performed with the IHC Toolbox plugin and the H-DAB model. The image was then converted to 8-bit format. Next, image correction was performed with the “uncalibrated” function. The average optical density value was determined, and the mean optical density (Mean) was calculated with the following equation: Mean = integrated optical density (IntDen)/area of the entire image (Area).

ChIP assays were carried out by ChIP Assay Kits (Thermo Fisher Scientific) with SKOV3 or OVCAR3 cells. For DNA-protein cross-links, SKOV3 or OVCAR3 cells were incubated with 1% formaldehyde for 10 min, and then an ultrasound machine was utilized to break the cross-linked chromatin DNAs into segments sized 200 to 1000 bp. The chromatin lysate was precipitated by anti-H3K27ac (Abcam), anti-CBP (Abcam) or anti-IgG (Abcam). Finally, RT-qPCR was performed to analyze the ChIP samples.

Transfected cells were lysed using RIPA, and thereafter, the collected lysates underwent immunoprecipitation using anti‐CBP, anti‐P300 or anti‐IgG (Abcam) for 1 hour, followed by the addition of Protein A‐agarose into lysates and the incubation for 12 hours. Then, complex of protein A‐agarose‐antigen‐antibody was collected through centrifugation at 12 000 g at 4°C for 2 minutes. After washing, precipitated proteins were tested by Western blot.