Anti hdac3

Brain tissue and in vitro cells were prepared as previously indicated (Sessa et al., 2019 (link)) for western blot analysis. The following primary antibodies were used: anti-TBL1XR1 (Novus Biological NB600-270); anti-NCOR1 (Merck-Millipore ABE251); anti-HDAC3 (Abcam ab 13704); anti-GAPDH (Abcam ab8245); anti-βCATENIN (Chemicon AB19022); anti-pβCATENIN (Ser33/37/Thr41, Cell signaling #9561S); anti-ERK1 (Cell Signaling Technology 4372); anti-pERK1 (Cell Signaling Technology 5726); Anti-V5 (Thermo Fisher Scientific R960-25); Anti-Histone H3 (Abcam, ab1791).

Protein was extracted from the fresh ACP surgical specimens by lysing cells with protease inhibitor cocktail (Roche, USA). After measuring the protein content with a Bradford assay, 20 μg of protein was resolved by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in TBST for 2 h at room temperature and incubated overnight with the following antibodies: anti-HAT, anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-HDAC8 (1:1000, Abcam, USA), anti-CBX4, anti-Runx2, anti-histone (1:1000, Cell Signaling Technology, USA), and anti-β-actin (1:1000, Proteintech, USA). The specimens were then incubated with secondary antibodies, IRDye800-conjugated anti-rabbit IgG and IRDye680-conjugated anti-mouse IgG (1:15,000, LiCor, USA), for 1 h at room temperature to label the primary antibody. An Odyssey Infrared Image System (LiCor, USA) was used to analyze signal intensities. The densitometry results were first normalized to the density obtained for β-actin or histone and then compared with that of the control to obtain relative fold changes.

ChIP assays were performed as previously described [24 (link)]. Anti-PU.1 (the same clone as that used for Western blotting analysis), anti-acetyl-histone H3 (#06–599, Millipore), goat IgG (#02–6202, Invitrogen), or rabbit IgG (#02–6102, Invitrogen) were used for immunoprecipitation. Anti-HDAC3 (#ab47237), anti-H3K4me3 (#ab8580), and their control rabbit IgG (#ab46540) were purchased from abcam (Cambridge, United Kingdom). Quantitative PCR of precipitated chromosomal DNA was performed using an Applied Biosystems StepOne real-time PCR system. The nucleotide sequences of the primer sets used for quantitative PCR targeting the GATA3 proximal promoter and CGRE on the IL-13 promoter are described in S1 Table.

Commercially available antibodies were used: anti-RORα (sc-28612; 1:1000 dilution for IB analysis, 5 μg for ChIP assay), anti-tubulin (sc-8035, 1:5000 dilution for IB analysis), anti-AKT (sc-8312; 1:1000 dilution for IB analysis), anti-PPARα (sc-9000x, 1 μg for ChIP assay) and anti-GFP (sc-9996, 1 μg for ChIP assay) from Santa Cruz Biotechnology; anti-β-actin (A5441; 1:5000 dilution for IB analysis) and anti-FLAG (F3165, Sigma, 1:5000 dilution for IB analysis, 1 μg for IP assay) from Sigma; anti-HA (MMS-101R; 1:5000 dilution for IB analysis, 1 μg for IP assay) from Covance; anti-H3Ac (#06-599, 1 μg for ChIP assay) from Millipore; anti-phospho-AKT(Ser473) (#4051 S, 1:1000 dilution for IB analysis) from Cell Signaling; anti-PPARγ (ab41928, 1 μg for ChIP assay), anti-PGC1α (ab54481, 1 μg for ChIP assay) and anti-HDAC3 (ab7030, 1 μg for ChIP assay) from Abcam; anti-RNA polymerase II (MMS-126R, 1 μg for ChIP assay) from Berkeley antibody company; anti-V5 (R96025; 1:5000 dilution for IB analysis) from Invitrogen.

RNA immunoprecipitation (RIP) assays were performed using the EZ-Magna RIP kit (Millipore) following the manufacturer’s instruction. Five micrograms of anti-EZH2 (Millipore) and anti-HDAC3 (Abcam) antibodies were used.

Primary antibodies used in this study were as follows: anti-Actin (Abcam, ab179467), anti-CDK1 (Abcam, ab133327), anti-STAT3 (Abcam, ab109085; Abcam, ab32500), anti-CD44 (Abcam, ab50137), anti-CD24 (Abcam, ab179821), anti-Sox2 (Cell Signailing Technology, #23064; Abcam, ab92494), anti-Oct4 (Abcam, ab181557; Cell Signailing Technology, #2890), anti-Nanog (Cell Signailing Technology, #4903), anti-HDAC3 (Abcam, ab32369), anti-Myc-Tag (Cell Signailing Technology, #2276), anti-Phospho-CDK1 (Tyr15) (Abcam, ab47594; Cell Signailing Technology, #4539), anti-Phospho-CDK1 (Thr14) (Cell Signailing Technology, #2543); anti-Phospho-CDK1 (Thr161) (Cell Signailing Technology, #9114); anti-Phosoho-STAT3 (Tyr705) (Abcam, ab76315; Affinity, AF3293), anti-Phospho-STAT3 (Ser727) (Abcam, ab32143), anti-Acetyl-lysine (PTM Bio, PTM-105; PTM Bio, PTM-101; Cell Signaling Technology, #9441S), anti-Ubiquitin (Abcam, ab134953); FITC-CD44 (BD Biosciences, catlog.555478), PE-CD24 (BD Biosciences, catlog.555428), Fisetin was purchased from Selleck (catalog.S2298). Trichostatin A (TSA) and Nicotinamide (NAM) was purchased from MCE (catalog. HY-15144; catalog. HY-B0150).