Anti cbp

Isoproterenol and Trichostatin A (TSA) were purchased from Sigma Aldrich (St. Louis, MO, USA) and used at 10 µM and 100 nM, respectively. Murine TNF-α was a gift from the VIB Department for Molecular Biomedical Research of Ghent University (VIB-UGent, Gent, Belgium) and was used at 2000 IU/ml. Insulin-like growth factor-1 (IGF-1) was from ImmunoTools (Friesoythe, Germany) and was used at 10 ng/ml. Anti-β₂-AR, anti-TNF-R1, anti-myogenin, anti-PARP, anti-P-H3-Ser10, anti-CBP, anti-RNA polymerase II, anti-p65, anti-IκBα, anti-P-CREB-Ser133 and anti-PKAc were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-P-p65-Ser536, anti-P-ERK-Thr202/Tyr204, anti-P-JNK-Thr183/Tyr185, anti-P-p38-Thr180/Tyr182, anti-P-MSK-1-Thr581, anti-Lamin A/C and anti-CREB were from Cell Signaling Technology (Danvers, MA, USA). Anti-Ac-H3-Lys27 and GAPDH were from AbCam (Cambridge, UK). Anti-α-tubulin and anti-α-actin were from Sigma-Aldrich. In figures, the expression “antibody anti-” was substituted by the Greek letter “α-”. AatII and HincII restriction enzymes were obtained from New England BioLabs (Ipswich, MA, USA).

Mice were sacrificed for histopathologic evaluation 14 weeks after irradiation. The skin and soft tissue of the irradiated leg was formalin-fixed, paraffin-embedded, cut into 4 μm sections, and stained with hematoxylin-eosin using standard procedures. To detect collagen accumulation in fibrotic tissues, Masson's trichrome staining was performed according to the manufacturer's protocol (Diagnostic BioSystems, Pleasanton, CA). For the immunohistochemical analysis, deparaffinized slides were incubated with anti-TGF-β1 (Cell Signaling), anti-PAI-1 (Santa Cruz Biotechnology), anti-SMA (abcam), anti-MMP2 (Santa Cruz Biotechnology), anti-MMP9 (Santa Cruz Biotechnology), anti-p300 (Santa Cruz Biotechnology), anti-CBP (Santa Cruz Biotechnology), and anti-acetylated p65 (Cell Signaling) primary antibodies, and subsequently with horseradish peroxidase-conjugated secondary antibodies. The DAB⁺ chromogen were used for signal detection (Dako Real Envision detection kit; Dako, Glostrup, Denmark). Representative images of the brown staining within fibrotic regions were captured and evaluated.

HuH7 cells were seeded on 100 mm dishes to approximately 60–70% confluence. After overnight incubation, cells were transfected with appropriate amount of expression plasmids using calcium phosphate coprecipitation method. Forty-eight hours later, nuclear extracts were prepared and co-IP was performed as described previously18 (link). Briefly, nuclear extracts (200 μg) were incubated with 20 μl anti-HA agarose beads (Sigma-Aldrich), anti-Flag M2 agarose resins (Sigma-Aldrich) or protein G Sepharose-conjugated rabbit anti-p300 (Santa Cruz) and anti-CBP (Santa Cruz) in co-IP buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100 and protease inhibitor) at 4 °C overnight. The beads with immunoprecipitates were rinsed thrice with cold PBS containing 0.5% NP40. The eluted complexes were subjected to SDS-PAGE followed by immunoblotting with antibodies against HA and Flag (Sigma-Aldrich).

ChIP experiments were performed, as previously described.^{32 (link)} Briefly, cells were synchronised with α-amanitin,^{33 (link)} then treated for 45 min with E2 (1 nM), ICI (10 nM), abiraterone (7.5 μM) or combination and fixed. The antibodies used were anti-ER (Santa Cruz Biotechnology; sc-543×), anti-CBP (Santa Cruz Biotechnology; sc-369×) and Mouse IgG1 (Dako, Denmark A/S). The resulting DNA was subjected to quantitative PCR analysis using SYBR green (Applied Biosystems) with the following primers for TFF1: (forward) 5′-GGC CAT CTC TCA CTA TGA ATC ACT TCT GCA-3′ and (reverse) 5′-GGC AGG CTC TGT TTG CTT AAA GAG CGT TAG-3′, and for GREB1: (forward) 5′-GAA GGG CAG AGC TGA TAA CG 3′ and (reverse) 5′-GAC CCA GTT GCC ACA CTT TT.

Protein extracts from cortical neurons or tissues were prepared with sample buffer [60 mM tris-HCl (pH 6.8], 2% sodium dodecyl sulfate, 10% glycerol, 5% 2-mercaptoethanol, and 0.1% bromophenol blue] and boiled for 5 min. For FACS sorting, the whole cortex dissected from WT or Fmr1 KO was homogenized by douncing followed by nuclei extraction via sucrose gradient ultracentrifugation (69 (link)). The nuclei were recovered from the pellet, resuspended, and incubated with NeuN antibody (Millipore, MAB377). Immunotagging with anti-NeuN conjugated to Alexa Fluor 488 (Invitrogen, A-21202) allows for sorting of the NeuN⁺ neuronal nuclei by fluorescence-activated sorting through a FACS machine (MoFlo Astrios, Beckman Coulter), followed by Western blotting. Proteins were separated by SDS-PAGE and subjected to Western analysis using the following antibodies: anti-BRD2 (Bethyl, A302-583A), anti-BRD3 (Active Motif, 61489) or anti-BRD4 (Bethyl, A301-985A100), anti-CBP (Santa Cruz, sc-369x), anti-FMRP (Cell Signaling, 4317S), anti–β-actin (Santa Cruz, sc-47778), anti–α-tubulin (Cell Signaling, 2144S), anti-H3K27ac (Abcam, Ab4729), anti-H4K8ac (Abcam, Ab15823), anti-H3K4me1 (Abcam, Ab8895), anti-H3ac (Millipore, 06-599), or anti-H3 (Abcam, ab176842).