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Ab56919

Manufactured by Abcam
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Sourced in United States, China

Ab56919 is a lab equipment product. It is a device used for the detection and quantification of specific proteins or other biomolecules in a sample.

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Lab products found in correlation

Ab4441, by Abcam (2 mentions) Mecamylamine, by Merck Group (2 mentions) Nifedipine, by Merck Group (2 mentions) Ab176334, by Cell Signaling Technology (2 mentions) Ab52084, by Abcam (2 mentions) Ab6046, by Merck Group (2 mentions) Nicotine hydrogen tartrate, by Merck Group (2 mentions) G8795 200ul, by Merck Group (2 mentions) Ab8580, by Abcam (2 mentions) Sc 899, by Santa Cruz Biotechnology (1 mentions) Sc 225, by Santa Cruz Biotechnology (1 mentions) Ab13665, by Abcam (1 mentions) Nuclear lysis buffer, by Merck Group (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Ez magna chip a g kit, by Merck Group (1 mentions) Anti rna pol 2, by Merck Group (1 mentions) Ab62341, by Merck Group (1 mentions) Alexa fluor dye, by Thermo Fisher Scientific (1 mentions) Ab5905, by Merck Group (1 mentions) Chicken anti gfp, by Thermo Fisher Scientific (1 mentions)

11 protocols using ab56919

1

ChIP Assay for Histone Modifications

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The ChIP assay was performed using the Magna-CHIP™ A chromatin immunoprecipitation kit (EMD Millipore), followed by PCR as previously described [67 (link)]. rabbit anti-IgG (Santa Cruz Biotechnology, sc-66931), rabbit anti-WDR5 ( Abcam, AB56919), or rabbit anti-acetyl-Histone H3 antibody (Abcam, AB4441) was used. The primers for amplifying the promoter regions from the precipitated fractions were 5′- AGTCAGGGGAGCAGCGGTGCG-3′ (ApoER forward), 5′-CCCCATCTCGGAGCCGCCTCT-3′ (ApoER reverse), 5′- GCACGCACGCTGCCACTTGCC-3′ (VldlR forward), 5′-GGGAACGGCGCGGGCGGCGGG-3′ (VldlR reverse), 5′- ACACCCAACCAGGTCAGAGCCG-3′ (CDK5 forward), and 5′-CCAGGAAGCATCGCGTGTCGGG-3′ (CDK5 reverse).
Ka M., Kim H.G, & Kim W.Y. (2022). WDR5-HOTTIP histone modifying complex regulates neural migration and dendrite polarity of pyramidal neurons via reelin signaling. Molecular neurobiology, 59(8), 5104-5120.
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2

Nicotine-Induced Cellular Signaling Assay

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Nicotine hydrogen tartrate (Sigma, St Louis, MO, USA) was dissolved in phosphate-buffered saline (PBS) pH 7.4 and placed at 20°C until use. Inhibitors were incubated with the cultures for the following durations before nicotine stimulation: mecamylamine (20 min; Sigma), α-bungarotoxin (α-BTX; 20 min; Sigma and Calbiochem) and nifedipine (15 min; Sigma). Most inhibitors were used at a stock concentration of 1000X of the desired stimulating concentration. Anti-Ash2l antibody (abcam, ab176334) and anti-Mef2c antibody (Cell Signaling, 5030) were validated using cells overexpressing Ash2l and Mef2c. Anti-Wdr5 antibody (Abcam, ab56919) and anti-Rbbp5 antibody (Abcam, ab52084) have been validated in published studies (supplementary methods checklist). Anti-β tubulin antibody (Abcam, ab6046) and anti-Gapdh antibody (Sigma, G8795-200UL) were used as loading controls for Western blots and have been validated in published studies (supplementary methods checklist).
Jung Y., Hsieh L.S., Lee A.M., Zhou Z., Coman D., Heath C.J., Hyder F., Mineur Y.S., Yuan Q., Goldman D., Bordey A, & Picciotto M.R. (2016). An epigenetic mechanism mediates developmental nicotine effects on neuronal structure and behavior. Nature neuroscience, 19(7), 905-914.
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3

Nuclear Protein Extraction and Western Blotting

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Nuclear extracts were prepared from hippocampal cells using a buffer that contains 420 mM NaCl, 25% glycerol, 0.2 mM EDTA, 1 mM DTT, 20 mM HEPES pH 7.9, 1.5 mM MgCl2, and protease inhibitors as reported previously (28 (link), 58 (link)). Proteins were detected by western blot using specific antibodies: monoclonal Ezh2 (AC22, Cell Signaling), polyclonal Ezh1 (ab13665, Abcam), monoclonal JBP1/PRMT5 (611539, BD Transduction Laboratories), monoclonal Wdr5 (ab56919, Abcam), polyclonal RNA Pol II (sc-899, Santa Cruz Biotechnology), polyclonal TFIIB (sc-225, Santa Cruz Biotechnology).
Aguilar R., Bustos F.J., Saez M., Rojas A., Allende M.L., van Wijnen A.J., van Zundert B, & Montecino M. (2016). Polycomb PRC2 complex mediates epigenetic silencing of a critical osteogenic master regulator in the hippocampus. Biochimica et biophysica acta, 1859(8), 1043-1055.
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4

Chromatin Immunoprecipitation of WDR5 and H3K4me3

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Cells were transfected with si-WDR5-2 or control siRNA for 72 h. Chromatin immunoprecipitation was conducted with the EZ-Magna ChIP A/G kit (Millipore) according to the manufacturer's instructions. Briefly, 1 × 106 cells were used for each reaction. Cells were fixed in 1% formaldehyde at room temperature for 10 min, the nucleus was isolated with nuclear lysis buffer (Millipore) supplemented with protease inhibitor cocktail (Millipore). Chromatin DNA was sonicated and sheared to a length between 200 bp and 1000 bp. The sheared chromatin was immunoprecipitated at 4°C overnight with anti-WDR5 (Abcam, ab56919) and anti-H3K4me3 (Abcam, ab8580) antibodies. Normal rabbit IgG was used as a negative control and the anti-RNA pol-II (Millipore) antibody was used as a positive control. The WDR5, H3K4me3 and RNA pol-II protein level in the ChIP assays were detected by Western blotting (Fig S6). The negative control primers were used to detect the DNA fragment out of genes as a distal region control. Primers for ChIP-qPCR are listed in Supplementary Table 3.
Chen X., Xie W., Gu P., Cai Q., Wang B., Xie Y., Dong W., He W., Zhong G., Lin T, & Huang J. (2015). Upregulated WDR5 promotes proliferation, self-renewal and chemoresistance in bladder cancer via mediating H3K4 trimethylation. Scientific Reports, 5, 8293.
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5

Immunostaining of Neural Cells

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Immunostaining of brain sections or dissociated neural cells was performed as described previously [57 (link),58 (link)]. The following primary antibodies were used: Mouse anti-WDR5 (Abcam, AB56919), and rabbit anti-Neu N (Abcam, AB 104225), rabbit anti-c-Myc (Abcam, AB39688), chicken anti-MAP2 (Abcam, AB5392), chicken anti-GFP (Thermo Fisher Scientific, A10262), rabbit anti-GFP (Thermo Fisher Scientific, A11122), rabbit anti-RFP (Abcam, AB62341), guinea pig anti-vGlut (EMD Millipore, AB5905), and rabbit anti-vGAT (EMD Millipore, AB5062P). Appropriate secondary antibodies conjugated with Alexa Fluor dyes (Thermo Fisher Scientific) were used to detect primary antibodies.
Ka M., Kim H.G, & Kim W.Y. (2022). WDR5-HOTTIP histone modifying complex regulates neural migration and dendrite polarity of pyramidal neurons via reelin signaling. Molecular neurobiology, 59(8), 5104-5120.
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6

Comprehensive Antibody Panel for Chromatin Immunoprecipitation

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The following antibodies were used for IP, chromatin immunoprecipitations (ChIP), and western blot: rabbit anti-MYC (N262, sc-764; C19, sc-788, both Santa Cruz; IG13, 022Y, 1236 all three provided by L.-G. Larsson), rabbit anti-MAX (C17, sc-197, Santa Cruz), rat anti-MXD1 (5F4), mouse anti-nucleolin (MS-3, sc-8031, Santa Cruz) rabbit anti-ASH2L (A300–489A, Bethyl; sera 548 and 549 (antigen ASH2L-1-444)), rat anti-ASH2L (4C5 and 4B5 (antigen ASH2L-1-279)), mouse anti-WDR5 (ab56919, abcam), rabbit anti-RbBP5 (A300-109A, Bethyl), rabbit anti-H3 (ab1791, abcam), rabbit anti-H3ac (06-599, Millipore), rabbit anti-H3K9ac (ab4441, abcam), rabbit anti-H3K27ac (ab4729, abcam), rabbit anti-H3K4me3 (ab8580, abcam), rabbit anti-H3K4me2 (07-030, Millipore), rabbit anti-H3K4me1 (ab8895, abcam), rabbit anti-H3K9me3 (ab8898, abcam), rabbit H3K9me2 (07-212, Millipore), rabbit anti-H3K9me1 (ab9045, abcam), rabbit anti-H3K27me3 (ab6002, abcam), rabbit anti-CBP (C-20, sc-583, Santa Cruz), rabbit anti-p300 (N-15, sc-584, Santa Cruz), rabbit IgG (Kch-504-250, Diagenode), mouse anti-actin (MP Biomedicals), rat anti-HA (3F10, Roche), mouse anti-Flag (M2, Sigma-Aldrich), rabbit anti-caspase 5 (2157, was kindly provided by A. Krippner-Heidenreich).
Ullius A., Lüscher-Firzlaff J., Costa I.G., Walsemann G., Forst A.H., Gusmao E.G., Kapelle K., Kleine H., Kremmer E., Vervoorts J, & Lüscher B. (2014). The interaction of MYC with the trithorax protein ASH2L promotes gene transcription by regulating H3K27 modification. Nucleic Acids Research, 42(11), 6901-6920.
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7

RIP Assays of Epigenetic Regulators

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RIP assays were performed using the EZ-Magna RIP Kit (Millipore). Briefly, 1 × 107 cells were lysed with RIP lysis buffer. Cell extracts were co-immunoprecipitated with Anti-TET1 antibody (Active motif, 61741, 2 µg per 107 cell lysate), Anti-KAT3B/p300 antibody (Abcam, ab10485, 2 µg per 107 cell lysate), Anti-WDR5 antibody (Abcam, ab56919, 2 µg per 107 cell lysate), and Anti-DOT1L Antibody (Thermo Fisher, A300-953A, 2 µg per 107 cell lysate). The recovered RNA was subjected to RT-qPCR analysis and U1 was used as a nonspecific control target.
Zhou T., Zhu X., Ye Z., Wang Y.F., Yao C., Xu N., Zhou M., Ma J., Qin Y., Shen Y., Tang Y., Yin Z., Xu H., Zhang Y., Zang X., Ding H., Yang W., Guo Y., Harley J.B., Namjou B., Kaufman K.M., Kottyan L.C., Weirauch M.T., Hou G, & Shen N. (2022). Lupus enhancer risk variant causes dysregulation of IRF8 through cooperative lncRNA and DNA methylation machinery. Nature Communications, 13, 1855.
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8

Western Blot Analysis of Nuclear Proteins

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Nuclear extracts were prepared as reported previously (Paredes et al., 2004 (link)). The protein levels were quantified by Bradford’s assay using bovine serum albumin as a standard. For western blot analyses, 10 μg of total protein was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membrane. Immunoblotting was performed with secondary antibodies conjugated to horseradish peroxide and enhanced chemiluminescence solutions (Perkin-Elmer, Boston, MA, USA). Primary antibodies used were the following: Gapdh (ab9485, Abcam, Cambridge, MA, USA), αTFIIB C-18 (sc-225, Santa Cruz Biotechnology, Dallas, TX, USA), RNA-PolII N-20 (sc-899, Santa Cruz Biotechnology), Runx2 S-19 (sc-12488, Santa Cruz Biotechnology), Wdr5 (ab56919, Abcam), JARID1B/KDM4B (ab50958, Abcam), Ezh2 (39901, Active Motif, Carlsbad, CA, USA), Utx/Kdm6A (ab91231, Abcam), Prmt5/Jbp1 (611539, BD Biosciences, San Jose, CA, USA).
Rojas A., Sepulveda H., Henriquez B., Aguilar R., Opazo T., Nardocci G., Bustos F., Lian J.B., Stein J.L., Stein G.S., van Zundert B., van Wijnen A.J., Allende M.L, & Montecino M. (2018). Mll-COMPASS complexes mediate H3K4me3 enrichment and transcription of the osteoblast master gene Runx2/p57 in osteoblasts. Journal of cellular physiology, 234(5), 6244-6253.
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9

Western Blot Analysis of Developmental Regulators

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Whole cell extract was prepared by lysing cells in NP-40 lysis buffer with proteinase inhibitors (Roche) and phosphatase inhibitor (Thermofisher). Protein concentration was determined by BCA assay (Pierce). Proteins were separated by SDS-PAGE, transferred to PVDF membrane and immunoblotted overnight at 4 o C with 1:1000 primary antibodies against HOXA13 (ab106503; abcam), MLL1 (#ABE240; Millipore), MLL2 (#ABE206, Millipore), MLL4 (NB600-254, Novus Biologicals), WDR5 (ab56919; abcam), CYB5R2 (H00051700-B02P; abnova), CYP26B1 (H00056603-M01; abnova), UCP2 (ab97931; abcam) and GAPDH (#5174; Cell signaling). Then, the blots were washed three times with TBST, followed by incubation with 1:2000 secondary anti-rabbit antibody or secondary anti-mouse antibody at room temperature for 1 hr.
Wong C.H., Li C.H., He Q., Chan S.L., Tong J.H., To K.F., Lin L.Z, & Chen Y. (2020). Ectopic HOTTIP expression induces noncanonical transactivation pathways to promote growth and invasiveness in pancreatic ductal adenocarcinoma. Cancer letters, 477.
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10

Immunoprecipitation of INO80 and Wdr5

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MSC were isolated in radioimmunoprecipitation assay buffer (RIPA buffer). For IP, the lysates were immunoprecipitated using protein A beads mixed with 2 μg of normal rabbit IgG, INO80 or Wdr5 antibody at 4 °C overnight. The beads were washed and eluted. Input and IP samples were resolved on a SDS-PAGE gel, transferred onto PVDF membrane, and immunoblotted with specific antibodies as shown in the figure legends. Antibodies information is listed as below: INO80 (1:1000, ab22238, Abcam, China), Wdr5 (1:1,000, ab56919, Abcam, China). All of the IP experiments were repeated at least three times.
Zhou C., Zou J., Zou S, & Li X. (2016). INO80 is Required for Osteogenic Differentiation of Human Mesenchymal Stem Cells. Scientific Reports, 6, 35924.
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