Ab188408

Manufactured by Abcam

Ab188408 is a mouse monoclonal antibody that recognizes human Bcl-2. Bcl-2 is a regulator of programmed cell death or apoptosis. The antibody can be used in various immunological techniques, such as Western blotting and immunohistochemistry, to detect and study the expression of Bcl-2 in biological samples.

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6 protocols using ab188408

Co-immunoprecipitation of Protein Complexes

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Co-immunoprecipitation (Co-IP) was performed as previously described [9 (link), 12 (link), 14 (link)], with antibodies specific for hnRNPU (ab10297), CTCF (ab188408), FLAG (ab125243), or Myc (ab9106, Abcam Inc.). Bead-bound proteins were released and analyzed by Western blot.

Song H., Li D., Wang X., Fang E., Yang F., Hu A., Wang J., Guo Y., Liu Y., Li H., Chen Y., Huang K., Zheng L, & Tong Q. (2020). RETRACTED ARTICLE: HNF4A-AS1/hnRNPU/CTCF axis as a therapeutic target for aerobic glycolysis and neuroblastoma progression. Journal of Hematology & Oncology, 13, 24.

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Western Blot Analysis of Cellular Proteins

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Tissue or cellular protein was extracted with 1 × cell lysis buffer (Promega, Madison, WI). Western blot was performed as previously described [9 (link), 11 (link)–15 (link)], with antibodies for HNF4A (ab181604), hexokinase 2 (HK2, ab104836), solute carrier family 2 member 1 (SLC2A1, ab40084), v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN, ab16898), hnRNPU (ab10297), CTCF (ab188408), clusterin (CLU, ab69644), C-X-C motif chemokine receptor 4 (CXCR4, ab124824), trophoblast glycoprotein (TPBG, ab129058), uveal autoantigen with coiled-coil domains and ankyrin repeats (UACA, ab99323), FLAG (ab125243), Myc (ab9106), glutathione S-transferase (GST, ab19256), histone H3 (ab5103), or β-actin (ab6276, Abcam Inc., Cambridge, MA).

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Multiparametric Immunofluorescence Imaging

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Cells were fixed in 4% Paraformaldehyde (PFA, Electron Microscopy Sciences) for 10 min at RT followed by 10 min of permeabilization using the following permeabilization buffer (100 mM Tris-HCl pH 7.4, 50 mM EDTA pH 8.0, 0.5 % Triton X-100). The following primary antibodies were incubated overnight: OCT3/4 (1:100, sc-5279, Santa Cruz Biotechnology), ZSCAN4 (1:2000, AB4340, Millipore Sigma), γH2AX (1:1000, 05-636, Millipore), CTCF (1:1000, ab188408, Abcam), Flag (1:500, F1804, Sigma Aldrich). Corresponding Alexa Fluor 488 Chicken anti-Rabbit IgG (H+L) (Thermo Fisher Scientific, Cat# 31431), Alexa Fluor 488 Goat anti-Mouse IgG (H+L) (Thermo Fisher Scientific, Cat# A-11001), Alexa Fluor 647 Chicken anti-Rabbit IgG (H+L) (Thermo Fisher Scientific, Cat# A-21443) or Alexa Fluor 647 Chicken anti-Mouse IgG (H+L) (Thermo Fisher Scientific, Cat# A-21463) secondary antibodies were used to reveal primary antibody binding (1:1000). For generating the plots shown in Extended Data 2b, image analysis was performed using a custom Python script. In brief, DAPI-stained nuclei were segmented using the StarDist deep-learning image segmentation⁵². Segmented nuclei ROIs were used to quantify total DAPI intensity and RFP mean intensity.

Olbrich T., Vega-Sendino M., Tillo D., Wu W., Zolnerowich N., Pavani R., Tran A.D., Domingo C.N., Franco M., Markiewicz-Potoczny M., Pegoraro G., FitzGerald P.C., Kruhlak M.J., Lazzerini-Denchi E., Nora E.P., Nussenzweig A, & Ruiz S. (2021). CTCF is a barrier for 2C-like reprogramming. Nature Communications, 12, 4856.

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Investigating CTCF Interaction in DRGs

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Immunoprecipitation was conducted according to the instructions provided in a Co-IP Kit (Roche). The L3/4/5 dorsal root ganglions (DRGs) were isolated from sham and SNI with mitochrondrial injection therapy (SNI + Mito) groups. Each Co-IP sample consisted of 18 pooled DRGs from 6 mice per group. The protein extracts from DRGs were prepared following the same procedure as described for Western blot analysis. To eliminate any non-specific binding, the samples were incubated with protein G-agarose on a rotator at 4 °C for 3 h. After centrifugation, the supernatant was transferred to fresh tubes and mixed with 5 µl of anti-γH2AX antibody (#ab81299, Abcam) for 1 h. Subsequently, a homogeneous suspension of protein G-agarose (50 µL) was added and left to incubate overnight at 4 °C on a rotator. Following centrifugation and removal of the supernatant, the beads were washed twice with lysis buffer 1, twice with lysis buffer 2, and once with lysis buffer 3. Protein sample buffer was then added before boiling the samples for 5 min. The presence of CTCF in immunoprecipitated proteins was detected using an anti-CTCF antibody (#ab188408, Abcam) through Western blot analysis.

Zhang Y., Xu T., Xie J., Wu H., Hu W, & Yuan X. (2024). MSC-derived mitochondria promote axonal regeneration via Atf3 gene up-regulation by ROS induced DNA double strand breaks at transcription initiation region. Cell Communication and Signaling : CCS, 22, 240.

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Chromatin Immunoprecipitation (ChIP) Protocol

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The manufacturer's instructions for the ChIP assay (Cell Signaling Technology, CST, USA) were followed. Cells were cultivated to 80-90% confluency in RPMI 1640 medium with 10% FBS. After 10 minutes of formaldehyde fixation, glycine was added to terminate the process. After that, cells were scraped off and harvested using centrifugation (2,000 g, 4°C, 5 minutes). The pellet was re-suspended in ChIP cell lysis buffer and incubated for 10 minutes on ice, being stirred by inverting every 3 minutes. The supernatant was then discarded. Micrococcal nuclease is used to fragment the pelleted nuclear material after centrifugation of the nuclei at 4°C, 2,000 rpm, for 5 minutes. The pellet was sonicated after being re-suspended in the ChIP buffer. Immunoprecipitations were performed by adding antibodies into digested chromatin overnight at 4°C. The samples were re-suspended in protein G magnetic beads and incubated for 2 hours. Beads were then washed with ChIP buffer. The DNA was purified using DNA purification spin columns. The enrichment of particular DNA was submitted to standard PCR. Antibodies used were as follows: anti-AKAP8 (Abcam, ab72196), anti-Poly II (Abcam, ab19346), anti-CTCF (Abcam, ab188408), and anti-H3K4me3 (Abcam, ab213224). The Supplementary Table displays the primer sequences that were used in this study.

Mobet Y., Wang H., Wei Q., Liu X., Yang D., Zhao H., Yang Y., Ngono Ngane R.A., Souopgui J., Xu J., Liu T, & Yi P. (2024). AKAP8 promotes ovarian cancer progression and antagonizes PARP inhibitor sensitivity through regulating hnRNPUL1 transcription. iScience, 27(5), 109744.

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CUT&RUN Analysis of Neural Crest Factors

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Neural crest cells were dissected from HH9 embryos (n=20 per CUT&RUN experiment). Cells were dissociated in Accumax for 20min at RT under mild agitation. CUT&RUN experiments were carried out as previously described (Rothstein and Simoes-Costa, 2020 (link)). Cells were immobilized on BioMag Plus Concanavalin A magnetic beads (Bangs Laboratories, BP531) and incubated with rabbit anti-LEF1 (Abcam, #ab137872), anti-CTNNBI (Abcam, #ab32572) or anti-CTCF (Abcam, #ab188408) antibody (1:50) overnight at 4°C. After washing away unbound antibody, protein A-MNase was added to a final concentration of 700ng/mL and incubated for 1h at 4°C. Cells were cooled to 0°C and CaCl2 was added to a final concentration of 2mM to activate the MNase enzyme. MNase digestion was performed for 45min and terminated by the addition of 2XSTOP buffer. The protein-DNA complexes were released by centrifugation and digested with proteinase K for 10 min at 70°C. DNA fragments were isolated via phenol-chloroform extraction and ethanol precipitation. Protein A-MNase and spike-in DNA were kindly provided by Dr. Steven Henikoff (Skene and Henikoff, 2017 ). To quantify LEF1 binding in a Wnt loss-of-function context, HH4 embryos were electroporated with Wnt1/4 combined morpholinos and cultured until HH9 when neural crest cells were dissected and samples processed following the protocol previously described.

Azambuja A.P, & Simoes-Costa M. (2021). The connectome of neural crest enhancers reveals regulatory features of signaling systems. Developmental cell, 56(9), 1268-1282.e6.

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