Control rabbit igg

Immunoprecipitation was performed on MDA MB 468 cells using the Abcam immunoprecipitation kit (cat. No. ab206996), according to manufacturer’s instructions. Briefly, non-denaturing lysis buffer was used to collect 300 μg of cell lysate was incubated overnight with 3 μg/ml of either control rabbit IgG (Santa Cruz, cat. No. sc-2027) or IKKε rabbit polyclonal antibody (Abcam, cat. No. ab7891). Antibody bound proteins were captured using protein A/G sepharose beads, eluted, and analyzed via western blot. Antibodies used for western blot detection were purchased from Sigma (IKKε, cat. No. I4907), Santa Cruz (IKKα, cat. No. sc-7606 and NIK cat. No. sc-8417).

HSCs, cultured on 0.4 or 25.6 kPa hydrogels, were harvested for ChIP assay, ChIP assay was conducted using an EZ-Magna-ChIP HiSens kit (Millipore, #17-10461) as previously reported13 (link). Briefly, HSCs fixed with 1% formaldehyde and scraped from the hydrogels were subjected to nuclear lysis to release cross-linked protein/DNAs. The nuclear extract was then subjected to sonication and immunoprecipitation with anti-FOXC2 antibody (Abcam, #ab5060) or control rabbit IgG (Santa Cruz Technology, #sc-2027). Precipitated DNA fragments containing the promotor of ACTA2 (α-SMA), CTGF, FN1 and Col1A1 were quantitated by qPCR with the pair of primers. JASPAR software was used to predict the FOXC2 binding with the promoter of HSCs activation markers24 (link). The primers for ChIP-qPCR were displayed in Table S2.

Mice were deeply anesthetized with an intraperitoneal injection of pentobarbital (0.2 mg/g body weight) and sacrificed by cutting right atrium. Then, the 4% paraformaldehyde fixative was perfused from the left ventricle. The fixed abdominal mammary glands of lactating and post-weaning mice were collected, dehydrated in alcohol, embedded in paraffin, and sliced into 3-μm-thick sections. Immunohistochemistry for Kir2.1 in mammary gland paraffin sections was performed using rabbit anti-Kir2.1 antibody (ab65796; Abcam) as described in the previous study [15 (link)]. Briefly, for antigen retrieval, the sections were heated at 105°C for 15 min in 20 mM Tris-HCl buffer (pH 8.0) after deparaffinization. The sections were incubated with anti-Kir2.1 antibody (1: 800) or control rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C, and then incubated with biotin-conjugated anti-Rabbit IgG Abs and horseradish peroxidase-conjugated streptavidin (Nichirei, Tokyo, Japan). For signal development, 3,3′-diaminobenzidine (DAB) tetrahydrochloride-H₂O₂ solution was used. The sections were counterstained with Mayer’s hematoxylin.

ChIP-Seq experiments were performed with 5 x 10⁷ cells per assay with 10 μg of either rabbit anti- EBF1 (EMD Millipore AB10523), RBP-jκ Abcam AB25949), histone H3K4me3 (EMD Millipore 07–473), H3K4me1 (Active Motif 39297), H3K27me3 (Active Motif 39155) antibodies or control rabbit IgG (Santa Cruz Biotechnology sc-2027). ChIP was performed by as described previously with some modifications [42 (link)]. Briefly, crosslinked Mutu I or LCL lysates were sonicated to achieve a DNA fragment length of ~100–500 bp, incubated overnight with antibody-coated Dynabeads protein A/G, then washed with ChIP-seq wash buffer (50 mM HEPES, pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate, 1x protease inhibitors) for 5 times, then washed once with 50 mM NaCl in TE buffer. Immunoprecipitated DNA was eluted with ChIP-seq elution buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS), reverse-crosslinked at 65°C, treated with RNase A (0.2 mg/ml) and proteinase K (0.2 mg/ml), purified with phenol and chloroform, then subjected to qPCR validation. Validated ChIP DNA was isolated by agarose gel purification, ligated to primers, and then subject to Illumina-based sequencing using manufacturers recommendations (Illumina).