Anti histone 3

Embryos were collected on grape agar plates, dechorionated for 2 min in 50% bleach, and fixed in methanol-heptane (1:1) for 5 min. The fixed embryos were stored in methanol at −20°C. Before immunostaining, the embryos were first rehydrated gradually (5 min each in 1:3, 1:1, and 3:1 PTA:methanol and then 10 min in PTA). PTA consisted of PBS supplemented with 0.1% Triton X-100, and 0.02% azide. The embryos were then blocked in PBTA (PTA plus 1% BSA) for 30 min and incubated with primary antibodies (1:100 in PBTA) for 1 h at room temperature or overnight at 4°C. The following primary antibodies were used: anti-Histone 3 (Abcam, ab1791), anti-H3K9me1 (Active Motif, 39249), anti-H3K9me2 (Active Motif, 39683 and 39375), anti-H3K9me3 (Active Motif, 39765; Millipore, clone CMA308), anti-H3K9ac (Abcam, ab10812), anti-H3K27ac (Abcam, ab4729), anti-H3K27me3 (Cell Signaling, 9733), and anti-H4ac (Millipore, 06-598). The embryos were washed three times for 5 min each in PBTA and incubated with the appropriate fluorescently labeled secondary antibodies (Molecular Probes) for 1 h in the dark at room temperature. They were then washed four times for 5 min each in PBTA and mounted in VectaShield mounting medium with DAPI (Vector Laboratories). For TALE-light stainings, GFP- or mCherry-tagged purified TALE-light protein (1:500) was included during the incubation with secondary antibodies.

The total protein from liver tissues and hepatocytes was extracted and homogenized in RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.). The concentrations of the extracted nuclear and cytoplasmic fractions were quantified using the bicinchoninic acid assay method. A total of 50 µg protein per sample was separated by SDS-PAGE (10%) and then transferred to a PVDF membrane, prior to blocking with 5% non-fat milk in 1X TBST, overnight at 4°C. The membrane was then incubated with anti-β-catenin (1:1,000; product no. ab32572), anti-β-actin (1:5,000; product no. ab8227), anti-histone 3 (1:2,000; product no. ab62642; all from Abcam) primary antibodies for 2 h at room temperature. After washing three times in 1X TBST, the membranes were incubated with the corresponding HRP-conjugated secondary antibody (1:5,000; cat. no. A6154-1 ml; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. The membranes were washed once more, exposed to ECL (Sangon Biotech Co., Ltd.), and then photographed by ChemiDoc™ XRS+ Imaging system (Bio-Rad Laboratories, Inc.). The protein expression levels were quantified using ImageJ software version 1.46 (National Institutes of Health). Histone 3 and β-actin were used as internal controls.