Anti phospho creb

Nuclear proteins were extracted using a nuclear protein extraction kit according to the manufacturer’s instructions (Beyotime Co., Nantong, China). Protein concentrations were determined with a bicinchoninic acid protein assay kit (Beyotime). Aliquots of cell lysates containing 20 μg protein were separated in 10% polyacrylamide gels and electrophoretically transferred to PVDF membranes (Millipore) using a Bio-Rad criterion blotter. Membranes were blocked in 5% non-fat dried milk in TBST at room temperature for 1 h and then incubated with either anti-phospho-CREB (1:3000 dilution; Millipore) or anti-CREB (1:2000 dilution; Millipore) at 4 °C overnight. The blots were then washed and incubated with horseradish peroxidase-labeled secondary antibodies (Sigma) at 37 °C for 1 h. Immunoreactive bands were detected with enhanced chemiluminescence Western blotting detection reagents (Beyotime). The blots were exposed to X-ray film for radiography of the bands and were digitally detected and measured using a LAS3000 Bioimage Analyzer (Fuji Photo Film, Tokyo, Japan).

Chromatin immunoprecipitation (ChIP) assays were performed as described previously (Kim, Lee, Kim, et al., 2016). The chromatins were sheared to 200–800 bp sizes using an EpiShear Probe Sonicator (Active Motif, Carlsbad, CA, USA). For immunoprecipitation, 10 μg of sheared chromatin was added to 2 μg of primary antibody, and 20 μl of protein G magnetic beads was used. The antibodies used were anti‐MeCP2 (3456S; Cell Signaling), antiphospho‐CREB (06‐519; Millipore), and nonimmune rabbit IgG (ab37415; Abcam). Immunoprecipitated DNA was used for quantitative real‐time PCR, as described above.

Frozen-stored mammary tissues and liver tissues harvested on day 14 of pregnancy (p14), day 1 of lactation (L1), and day 10 of lactation (L10) were ground into powder with a mortar and pestle. The chromatin was fixed with 1% formaldehyde at room temperature for 10 min, and the fixation was quenched with glycine at a final concentration of 125 mM. The samples were processed as previously described. The following antibodies were used for ChIP-seq: anti-STAT5 (Santa Cruz, sc-835 and sc-271542), anti-phospho-CREB (Millipore, CS204400), anti-H3K27ac (Abcam, ab4729), anti-H3K4me3 (Millipore, 07-473), and anti-RNA Polymerase II (Abcam, ab5408). Libraries for next-generation sequencing (NGS) were prepared as previously described and sequenced with HiSeq 2500 (Illumina).
Quality filtering and alignment of the raw reads was performed using trimmomatic^{7 (link)} (version 0.36) and Bowtie^{8 (link)} (version 1.1.2), with the parameter m1 selected to retain only uniquely mapped reads, using the mm10 reference genome. Picard tools (Broad Institute. Picard, http://broadinstitute.github.io/picard/. 2016) were used to remove duplicates, and subsequently, Homer^{9 (link)} (version 4.8.2) software was applied to generate bedGraph files. The integrative Genomics Viewer^{10 (link)} (version 2.3.81) was used for visualization. Each ChIP-seq experiment was conducted with two replicates.