Rabbit anti‐KDM2B (09‐864; Millipore), anti‐EZH2 (ab191250 and ab191080; Abcam), anti‐LaminB1 (ab16048; Abcam), and goat‐anti rabbit HRP (CST) antibodies were used for western blots or immunohistochemistry (IHC). H3K36me2 (ab9049; Abcam) and RNA pol II (ab193468; Abcam) antibodies were used in a ChIP assay. Real‐time PCR primers and oligos used for shRNA cloning are listed in Table 1 . The oligos of hsa‐let‐7b‐5p‐inhibition(21257‐1)‐a and hsa‐let‐7b‐5p‐inhibition(21257‐1)‐b were used for cloning the let‐7b inhibitor sequence into the GV280 vector, and the oligos of hsa‐let‐7b(16144‐1)‐P1 and hsa‐let‐7b(16144‐1)‐P2 were used for cloning the let‐7b mimic sequence into the GV369 vector. Primers of KDM2B(21416‐11)‐P1 and KDM2B(21416‐11)‐P2 were used to amplify KDM2B coding sequences, and the 4062bp PCR product was cloned into the Age I/Age I site of GV358 vector. The sequences of shKDM2B, shEZH2, and shNC are as follows: TTCTTCAAACGCTGTGGAA, GAAATCTTAAACCAAGAAT, and TTCTCCGAACGTGTCACGT, respectively.
Ab9049
Manufactured by Abcam
Sourced in United Kingdom
Ab9049 is a primary antibody used in immunological techniques. It recognizes a specific target and can be utilized in applications such as Western blotting, immunohistochemistry, and ELISA. The core function of this product is to provide a tool for researchers to detect and study the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Ab9050, by Abcam (15 mentions)
Ab9048, by Abcam (8 mentions)
Ab1791, by Abcam (8 mentions)
Ab16048, by Abcam (3 mentions)
Ab8580, by Abcam (3 mentions)
Ab8898, by Abcam (3 mentions)
Ab7315, by Abcam (2 mentions)
Sinefungin, by Merck Group (2 mentions)
Actin a2066, by Merck Group (2 mentions)
Gapdh sc 166574, by Santa Cruz Biotechnology (2 mentions)
R pfi 2, by Merck Group (2 mentions)
Ab9045, by Abcam (2 mentions)
Ab1220, by Abcam (2 mentions)
Ab6002, by Abcam (2 mentions)
Ab24684, by Abcam (2 mentions)
Ab31739, by Abcam (2 mentions)
Ab113671, by Abcam (2 mentions)
H3k4me3, by Merck Group (2 mentions)
Ab9050, by Cell Signaling Technology (2 mentions)
F1804, by Merck Group (2 mentions)
31 protocols using ab9049
1
Comprehensive Epigenetic Regulatory Analysis
2
Histone Modifications in Gametocytes
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Gametocytes were treated with JIB-04 (5 µM) on days 2, 3 and 4 of development and sampled 24 h later. Histones were extracted as described before [17 (link)] with minor modifications. Nuclei were extracted by Dounce homogenisation in hypotonic lysis buffer (10 mM Tris–HCl (pH 8), 0.25 M sucrose, 3 mM MgCl2, 0.2% (v/v) Nonidet P-40) and protease inhibitors (Roche)). Histones were isolated from nuclei resuspended in Tris buffer (10 mM Tris (pH 8.0), 0.8 M NaCl, 1 mM EDTA and protease inhibitors) by overnight acid-extraction (0.25 M HCl, with rotation at 4 °C) and subsequent precipitated with 20% TCA. Histone pellets were washed with acetone, reconstituted in dddH2O and spotted quantitatively (100 ng per sample) on nitrocellulose membranes. Membranes were submerged in blocking buffer for 30 min followed by 1 h incubation with α-H3K36me2 (Abcam, ab9049) or α-H3K36me3 (Abcam, ab9050) primary antibody dilutions (1:5000 in TBS-t). Membranes were washed three times in TBS-t and then incubated with goat α-rabbit IgG antibody conjugated to HRP (1:10000) for 1 h. Chemiluminescent signal (Pierce SuperSignal West Pico PLUS Chemiluminescent Substrate) was quantified with ImageJ analysis software [95 (link)].
3
Antibody-based protein detection protocol
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Antibodies against SRF (sc-335), MyoD (sc-304), Set7/9 (sc-390823), MCK (sc-15164), skeletal α-actin (sc-58671), and GAPDH (sc-166574) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Flag (F1804), HA (H9658), GFP (G1544), and actin (A2066) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against histone H3 (tri-methylated K4, ab8580), histone H3 (dimethylated K36, ab9049), pan-methylated lysine (ab7315), and lamin B1 (ab16048) were obtained from Abcam (Cambridge, UK). Anti-KDM2B/JHDM1B antibody (09–864) was obtained from EMD Millipore Corp. (Billerica, MA, USA). Anti-MHC antibody (MF-20) was obtained from DSHB (Iowa City, IA, USA). Anti-V5 antibody (46–0705) was obtained from Invitrogen (Carlsbad, CA, USA). SRF-derived peptides (1~5, K154, K163, K165, and K165A) were synthesized by Peptron (Daejeon, South Korea). Sinefungin and (R)-PFI-2 were purchased from Sigma-Aldrich (St. Louis, MO, USA).
4
Validation of H3K36me2/3 Antibody Specificity
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The specificity of commercially obtained ChIP-grade rabbit anti-H3K36me2 (Abcam, ab9049) and anti-H3K36me3 (Abcam, ab9050) antibodies (same batch numbers as previously validated for specificity against H3K36me2&3, [22 (link), 34 (link), 39 (link)–41 (link), 43 (link)]) was tested using modified and unmodified synthetic peptides (Genscript) of the P. falciparum 3D7 histone H3 sequence as per ENCODE standards (www.encodeproject.org ; Additional file 4 : Table S8). Modified peptides were di- or tri-methylated on K9 or K36 each with a corresponding unmodified K9 (ARTKQTARKSTAGKAPRKQ) and K36 (ARKSAPISAGIKKPHRYRPG) peptide. Nitrocellulose membranes spotted with 25 ng of each peptide were blocked for 30 min in blocking buffer (5% milk powder in TBS-t (50 mM Tris (pH 7.5), 150 mM NaCl and 0.1% (v/v) tween-20) and incubated with primary antibodies (1:5000) against H3K36me2 or H3K36me3 overnight followed 1 h incubation with secondary antibody (1:10,000 goat anti-rabbit IgG conjugated to HRP). Chemiluminescent signal (Pierce Super Signal West Pico PLUS Chemiluminescent Substrate) was quantified by densitometry using ImageJ analysis software [95 (link)].
5
ChIP-seq analysis of H3K27 and H3K36 methylation
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H3K27me2/3 ChIP using α-H3K27me2/3 antibody (Active Motif, 39536), which recognizes di- or trimethylated H3K27, was performed as previously described (Wiles et al., 2020 (link)). H3K36me2 and H3K36me3 ChIP using α-H3K36me2 (Abcam, ab9049) and α-H3K36me3 (Abcam, ab9050) antibodies were performed as previously described (Bicocca et al., 2018 (link)). The isolated DNA was used for qPCR (see Key resources table for primers) or prepared for sequencing (Wiles et al., 2020 (link)). Sequencing was performed by the University of Oregon Genomics and Cell Characterization Core Facility.
6
Epigenetic Profiling of Drosophila Larval Brains
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Fly larval brains (w1118 control) were dissected and fixed for 10 min in 4% paraformaldehyde 1xPBS and blocked in PBST for 40 min. Rabbit anti-H3K36me1 (Abcam, Cambridge, UK; ab9048), anti-H3K36me2 (Abcam; ab9049), and anti-H3K36me3 (Abcam; ab9050) were each incubated at 1:200 dilution overnight at 4 °C, followed by incubation with Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at 1:1000 dilution for 2 h at room temperature. Confocal imaging was performed using a ZEISS ApoTome.2 microscope with 63× Plan-Apochromat 1.4 N.A. oil objective. Images were acquired using ZEN blue edition (version 3.0). ImageJ software (version 1.49) was used to process the images.
7
Histone Modification Antibody Protocol
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8
Comprehensive Protein Detection and Histone Modification Analysis
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To identify histone modifications, acid extraction of histone was performed as previously reported [48 (link)]. To detect other proteins, cells were extracted with lysis buffer (50 mM Tris-HCl, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1.5 mM PMSF and protease inhibitors cocktail). Equal amounts of protein were size fractionated on 6.0 to 15.0% SDS-PAGE gel. The antibodies used were anti-COX-2 (sc-19999, Santa Cruz), anti-c-Fos (ab7963, Abcam), anti-c-Jun (sc-44, Santa Cruz), anti-KMT3A (ab31358, Abcam), anti-KMT3B (17-10264, Merck Millipore), anti-KDM2A (ab31739, Abcam), anti-KDM2B (ab108276, Abcam), anti-KDM4A (ab70786, Abcam), anti-KDM4B (ab80473, Abcam), anti-NF-κB (sc-372G, Santa Cruz), anti-p300 (H-272, Santa Cruz), anti-CEBPβ (sc-150, Santa Cruz), anti-H3K4me1/2/3 (ab8895/ab7766/ab1012, Abcam), anti-H3K9me1/2/3 (ab9045/ab1220/ab8898, Abcam), anti-H3K27me1/2/3 (ab113671/ab24684/ab6002, Abcam), anti-H3K36me1/2/3 (ab9048/ab9049/ab9050, Abcam), anti-H3K79me1/2/3 (ab2886/ab3594/ab2621, Abcam), anti-H4K20me1/3 (ab9051/9053, Abcam), anti-histone H3 (ab131711, Abcam) and anti-β-actin (sc-1615, Santa Cruz).
9
ChIP-qPCR of H3K36me2 in Drosophila Imaginal Discs
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Chromatin preparation from hand-dissected haltere and third-leg imaginal discs (T3 discs) from wild-type or ash122 homozygous larvae and ChIP analysis was performed as described (Laprell et al., 2017 (link)). For each biological replicate, chromatin prepared from the discs of 60 larvae were used as input material. ChIP was performed with polyclonal anti-H3K36me2 antibody (Abcam ab9049) and qPCR primers used for analysis are listed in Table S5 .
10
ChIP-qPCR for Histone Modification
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ChIP-qPCR was performed as described before [16 (link)]. Briefly, ESCs were harvested and crosslinked with 1% formaldehyde, and cell fixation was ceased with the addition of glycine. The cells were primarily lysed, and chromatin extracts were collected and sonicated for obtaining soluble chromatin fragments. The chromatin samples were incubated with specific antibody and immunoprecipitated on protein G magnetic beads (GenScript, the USA). The immunoprecipitated DNA was next eluted, decrosslinked, and analyzed by qPCR. For immunoprecipitation, the antibody used was anti-H3K36me2 (ab9049, Abcam).
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