Sc 11417

Dewaxed hydrated paraffin-embedded tissue sections were immersed in 3% H₂O₂ and 100% methanol for 30 min at room temperature to quench endogenous peroxidase, and then the sections were blocked with 10% normal goat (for anti-HDAC3, Ki-67, ESR1, PGR, vimentin, and COUP-TFII antibodies) or rabbit (for anti-COL1 antibody) serum in PBS (pH 7.5) and incubated with anti-HDAC3 (1:1000 dilution, sc11417, Santa Cruz Biotechnology), anti–Ki-67 (1:1000 dilution, ab15580, Abcam), anti-ESR1 (1:500 dilution, M7047, DAKO), anti-PGR (1:500 dilution, A0098, DAKO), anti-vimentin (1:10,000 dilution, ab92547, Abcam), anti–COUP-TFII (1:500 dilution, PP-H7147–00, Perseus Proteomics), or anti-COL1 (1:1000 dilution, 1310–01, SouthernBiotech) antibodies overnight at 4°C. On the following day, the sections were incubated with secondary antibody conjugated to horseradish peroxidase (Vector Laboratories) for 1 hour at room temperature. Immunoreactivity was detected using diaminobenzidine (DAB; Vector Laboratories) and analyzed using microscopy software from NIS Elements Inc. (Nikon). The H-score was calculated using the following equation: H-score = Σ Pi (i), where i is the intensity of staining with a value of 1, 2, or 3 (weak, moderate, or strong, respectively) and Pi is the percentage of stained cells for each intensity, varying from 0 to 100%.

ChIP assays were performed as described previously with minor modifications^{12 (link)}. Briefly, purified rat SCs (~20 million cells) grown in the proliferation or differentiation (9 h in 1 mM cAMP-containing medium) condition were fixed for 10 min at room temperature with 1% formaldehyde–containing medium. Nuclei were isolated and sonicated in sonication buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA; 0.5 mM EGTA; and protease inhibitor cocktail). Sonicated chromatin (~300 μg) was used for immunoprecipitation via incubation with antibodies overnight at 4 °C. Prerinsed protein A/G PLUS-agarose beads (50 μl) were added to each ChIP reaction and incubated for 1 h at 4 °C. The beads were then incubated in 200 μl elution buffer at 65 °C for 20 min to elute immunoprecipitated materials. The ChIP–seq libraries were prepared using the NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (NEB, E6240L) and then were run on the Illumina sequencer HiSeq 2000. Two ChIP–seq replicates were performed for diff_HDAC3, p300, and IgG, and one replicate was performed for Pro_HDAC3, siHdac3_ H3K27ac, Scr_H3K27ac, H3k27ac, and H3K4me1. Antibodies against the following proteins were used: HDAC3 (Santa Cruz Biotechnology, sc-11417), p300 (rabbit; Santa Cruz, sc-585), H3K27ac (rabbit; Abcam, ab4729), and H3K4me1 (rabbit; Abcam, ab8895).

The protein level of CREBBP and HDAC3 in whole cell extracts were
detected by western blotting using rabbit anti-CREBBP (Santa Cruz Biotechnology,
sc-369) and rabbit anti-HDAC3 (Santa Cruz Biotechnology, sc-11417) respective.
Levels of H3K27ac were detected by western blotting using histone extracts
collected using the acid extraction method and blotted with Rabbit anti-H3K27ac
(abcam, ab4729) and Rabbit anti-total H3 (abcam, ab1791).

Membranes were blocked with casein (0.5%, v/v) before exposure to anti-HDAC3 (1:1000 dilution, sc11417, Santa Cruz Biotechnology), anti-PGR (1:1000 dilution, sc7208, Santa Cruz Biotechnology), anti-HDAC1 (1:1000 dilution, sc7872, Santa Cruz Biotechnology), anti-HDAC2 (1:1000 dilution, sc7899, Santa Cruz Biotechnology), or anti-β-actin (1:2000 dilution, sc1616, Santa Cruz Biotechnology) antibodies.