Anti ctcf

Mice were deeply anaesthetized and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). Then, the brains were removed and postfixed in the same fixative for 2 h at 4 °C and subsequently cryoprotected in 30% sucrose in PB. Frozen sections, either 10 or 50 μm thick, were prepared on a microtome. Sections were washed with PBS and incubated for 1 h at room temperature in blocking buffer: 20% Block Ace (KAC Co., Ltd.), 5% normal goat serum (NGS), 0.1% Triton X-100, 0.1% azide in PBS. Then, the sections were incubated overnight with primary antibody in antibody dilution buffer (5% Block Ace, 5% NGS, 0.1% Triton X-100, 0.1% azide in PBS) at 4 °C. Sections were then washed with 0.1% Triton X-100 in PBS and incubated for 1 h with secondary antibody in antibody dilution buffer at room temperature. The antibodies used were as follows: anti-calbindin (1:500; Sigma-Aldrich), anti-CTCF (1:1000; Cell Signaling Technology), anti-VGluT2 (1:10,000; Millipore), anti-active caspase-3 (1:500; Cell Signaling Technology), anti-calnexin (1:500; Enzo), anti-KDEL (1:2000; MBL), and anti-IP3R (1:500; abcam). For haematoxylin and eosin (HE) staining, sections were stained with Mayer’s haematoxylin and eosin Y (Muto Pure Chemicals, Tokyo, Japan).

Anti-CTCF (1:1000; Millipore, 07-729), anti-SMC1A (1:1000: Bethyl, A300-055A), anti-SMC3 (1:1,000; Abcam, ab9263), anti-RAD21 (1:1000; Abcam, ab992), anti-FLAG (1:1000; Sigma-Aldrich, F1804), anti-NUP153 (1:1000; Abcam, ab24700), anti-GAPDH (1:10,000; Sigma-Aldrich, G9545), anti-Histone H3 (1:10,000; Abcam, ab1791), and anti-α-TUBULIN (1:11,000; Santa Cruz, sc-5286) were used in western blot analysis. Note that anti-NUP153 (Abcam, ab24700) can also detect NUP62 and was used to detect NUP62 by western blot analysis. Anti-Rpb1 NTD (3 µl, Cell Signaling, 14958), Anti-CTCF (3 µl, Cell Signaling, 2899S), and anti-SMC3 (3 µg, Abcam, ab9263) were used in ChIP. Anti-LAMIN B1 (1:450; Abcam, ab16048), anti-V5 (1:400; Thermo Fisher Scientific, R960-25), anti-FLAG M2 (1:250; Sigma-Aldrich, F1804), anti-IgG(H+L)-Alexa555 (1:500; Thermo Fisher Scientific, A-21427), and anti-IgG(H+L)-Alexa488 (1:400; Thermo Fisher Scientific, A-11008 and A-32723) were used in immunofluorescence.

Chromatin immunoprecipitation (ChIP) was performed as previously described. Briefly, SKOV3 cells were rinsed with room temperature PBS before being crosslinked in a 1%formaldehyde solution. SKOV3 Cells were then harvested and homogenized in the presence of protease inhibitors before DNA was sonicated. Magnetic Dynal beads (Invitrogen, San Diego, CA, USA) combined with a mixture of antibodies (anti-CTCF, Cell Signaling Technology) was used to pull down CTCF overnight. Preimmune serum or IgG were used as negative controls. Sequencing libraries were generated for massive parallel sequencing using standard methods. Briefly, 500 ng of pulldown DNA was subjected to end repair, terminal adenylation, and adapter ligation before fragments ranging from, 400–500bp were isolated from a 2% E-gel (Invitrogen). After a standardized 12 cycle PCR, DNA quality was evaluated on a DNA 1000 Bioanalyzer chip (Agilent Technologies, Santa Clara, CA, USA) before being submitted for sequencing on an Illumina GAII (Illumina, Inc., San Diego, CA, USA). All ChIP-seq data is deposited in the Gene Expression Omnibus (GEO) database at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE8545) and their accession number is: GSE85453.