Rabbit or mouse igg

Localization of TRPV6, PMCA1, CTR1, ATP7A, IREG1, and HEPH proteins was examined by immunohistochemistry. Mouse placenta tissues were embedded in paraffin, cut into sections (4 μm thick), deparaffinized in xylene, and hydrated in descending grades of ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in TBS-T for 30 min. Nonspecific reactions were blocked by incubating the sections in 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were subsequently incubated overnight at room temperature with antibodies against TRPV6 (1:300, Santa Cruz Biotechnology), PMCA1 (1:300, Swant), CTR1 (1:300, Novusbio), ATP7A (1:300, Santa Cruz Biotechnology), IREG1 (1:300, Abcam), or HEPH (1:300, Abcam) diluted in 1% BSA. After washing with TBS-T, the sections were incubated with a biotinylated secondary antibody (rabbit or mouse IgG, Vector Laboratories) for 1 h at 37 °C and then incubated with ABC Elite (Vector Laboratories) for 30 min at 37 °C. Diaminobenzidine (DAB; Sigma-Aldrich) was used as a chromogen. The sections were counterstained with hematoxylin and mounted in Cytoseal* 60 Mounting Medium (Richard-Allan Scientific Co., Kalamazoo, MI, USA).

ChIP-PCR assays were performed by Simple ChIP plus Enzymatic Chromatin IP Kit (Cell Signaling: #9005) according to the manufacturer’s protocol. Briefly, cells were cross-linked with 1% of formaldehyde and lysed for nuclei preparation. Nucleus pellet was treated with Micrococcal nuclease and sonicated for chromatin fragmentation. Protein–chromatin complex was incubated with antibodies targeting anti-NFATc2 (ab2722), anti-NFKB1 (ab7971), anti-RELA (ab7970), anti-DNMT3A (ab13888), anti-DNMT3B (ab13604), anti-H3K4me1 (ab8895), anti-H3K4me3 (ab8580), anti-H3K9me3 (ab8898), anti-Ets1 (Cell Signaling: #14069), anti-H3Ac (Merck: 06–599), or anti-H3K27me3 (Merck: 07–449) at 4 °C for overnight. Rabbit or mouse IgG (Vector Laboratories) was used as negative control. After immunoprecipitation, 50 μl of Dynabeads protein G or A (Life technologies, Oslo) were added and rotated further for 6 h at 4 °C. Ab/protein/chromatin complexes were reverse-cross-linked at 65 °C overnight and DNA was purified by DNA purification columns (Cell Signaling: #10010). The relative enrichment of specific regions of precipitated DNA were measured by real-time PCR (qPCR). To quantify protein binding in specific genomic locus, purified DNA was used for real-time PCR. Primer sequences are listed in Supplementary Table 2. H3K27Ac, and H3K27me3 ChIP-seq data set (GSE38548) were re-analyzed^{35 (link)}.