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Anti wdr5

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Anti-WDR5 is a lab equipment product that functions as an inhibitor for the WDR5 protein. WDR5 is a key component of several chromatin-modifying complexes and plays a role in gene regulation. The Anti-WDR5 product provides a tool for researchers to study the function of WDR5 in various biological processes.

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Lab products found in correlation

Anti flag, by Merck Group (4 mentions) Anti rbbp5, by Fortis Life Sciences (3 mentions) Anti ash2l, by Fortis Life Sciences (3 mentions) Anti ha, by Abcam (3 mentions) Precast gel, by Bio-Rad (2 mentions) Anti tubulin, by Cell Signaling Technology (2 mentions) Histone extraction kit, by Abcam (2 mentions) 0.45 μm pvdf membrane, by Merck Group (2 mentions) Anti p53, by Cell Signaling Technology (2 mentions) Anti h3k4me2, by Merck Group (2 mentions) Anti h3k4me1, by Merck Group (2 mentions) Anti phospho p53 ser15 antibody, by Cell Signaling Technology (2 mentions) Wdr5 antibody, by Fortis Life Sciences (2 mentions) Anti phospho p53 ser392 antibody, by Abcam (2 mentions) Anti acetyl p53 k305 antibody, by Abcam (2 mentions) Anti actyl p53 k379 antibody, by Cell Signaling Technology (2 mentions) Anti h3k4me3, by Abcam (2 mentions) Anti wdr5, by R&D Systems (2 mentions) Anti h3, by Abcam (2 mentions) Anti β actin, by Cell Signaling Technology (2 mentions)

9 protocols using anti wdr5

1

Antibody Panel for Cell Signaling Analysis

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Antibodies used in this study: anti‐ASH2L (A300‐107A; Bethyl Laboratories), anti‐ASH2L (12331‐1‐AP; Proteintech Group), anti‐FLAG (4110‐FG; GNI), anti‐ERα (D8H8) (#8664; Cell Signaling Technology), anti‐ERα (F10) (sc‐8002; Santa Cruz Biotechnology), anti‐MLL1 (A300‐37A; Bethyl Laboratories), anti‐WDR5 (A302‐429A; Bethyl Laboratories), anti‐PAX2 (TA327502S; OriGene Technologies), anti‐Cyclin D1 (60186‐1‐lg; Proteintech Group), anti‐GAPDH (AC033; ABclonal Technology), anti‐Ki67 (sc‐15402; Santa Cruz Biotechnology), anti‐trimethyl H3‐K27 (07‐449; Millipore), anti‐trimethyl H3‐K4 (05‐745R; Millipore).
Zeng K., Wu Y., Wang C., Wang S., Sun H., Zou R., Sun G., Song H., Liu W., Sun N., Wei S., Liu W., Su Y., Zhou T., Zhang Y, & Zhao Y. (2020). ASH2L is involved in promotion of endometrial cancer progression via upregulation of PAX2 transcription. Cancer Science, 111(6), 2062-2077.
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2

Chromatin Immunoprecipitation (ChIP) Assay

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Antibodies used included anti-Flag (Sigma, F3165-5MG, clone M2), anti-MLL1C (Bethyl, A300-374A), anti-RbBP5 (Bethyl, A300-109A), anti-ASH2L (Bethyl, A300-112A), anti-WDR5 (Bethyl, A302-430A), anti-GFP (Clontech, 632377, clone JL-8), anti-trimethyl-Histone H3 (Lys4) (EMD Millipore, 07-473), anti-LANA LN53 (Millipore, MABE1109), goat anti-rat IgG (H + L) Alexa Fluor 488 (Invitrogen A11006), goat anti-rabbit IgG (H + L) Alexa Fluor 647 (Invitrogen A21245), human anti -LANA sera adsorbed against uninfected cell extract for western blot or affinity purified against carboxy-terminal LANA for ChIP, Anti-T7 tag® antibody (Abcam, ab9138), and anti-alpha-tubulin (Sigma T9026, cline DM1A).
Tan M., Li S., Juillard F., Chitas R., Custódio T.F., Xue H., Szymula A., Sun Q., Liu B., Álvarez Á.L., Chen S., Huang J., Simas J.P., McVey C.E, & Kaye K.M. (2021). MLL1 is regulated by KSHV LANA and is important for virus latency. Nucleic Acids Research, 49(22), 12895-12911.
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3

Histone Extraction and Immunoprecipitation from Embryonic Stem Cells

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ESCs or EBs were lysed with RIPA buffer (Pierce) in the presence of EDTA free protease inhibitor cocktail (Sigma). Histone extraction on EBs was performed using a histone extraction kit (Abcam) as described previously (Khan et al., 2015 (link)). Protein concentration was determined using Pierce BCA protein assay kit (ThermoFisher Scientific). 1.0 μg of histone extracts, or 10 μg of whole cell lysate was resolved on 4%–20% precast gel (Bio-Rad) was transferred to 0.45 μm PVDF membrane (Millipore). For immunoprecipitation, day 2 WT and Wdr5KO EBs were harvested and lysed in RIPA buffer with EDTA free protease inhibitor cocktail (Sigma). 1 mg sonicated cell lysates were subjected to immunoprecipitation using WDR5 antibody (Bethyl). The following primary antibodies were used for probing: anti-HA (1:10,000, Abcam, ab9110), anti-WDR5 (1:5000, R&D), anti-WDR5 (1:5,000, Bethyl), anti-Flag (1:2,000, Sigma), anti-H3 (1:10,000, Abcam), anti-H3K4Me1 (1:5,000, Millipore), anti-H3K4Me2 (1:10,000, Millipore), anti-H3K4Me3 (1:10,000, Abcam), anti-P53 (1:5,000, Cell Signaling), anti-Tubulin (1:10,000, Cell signaling), anti-β-Actin (1:10,000, Cell signaling), anti-Phospho-p53 (Ser15) Antibody (1:5,000, Cell Signaling), anti-Phospho-p53 (Ser392) Antibody (1:5,000, Abcam), anti-Acetyl-p53 (K305) Antibody (1:5,000, Abcam), anti-Actyl-p53 (K379) Antibody (1:5,000, Cell Signaling).
Li Q., Mao F., Zhou B., Huang Y., Zou Z., denDekker A.D., Xu J., Hou S., Liu J., Dou Y, & Rao R.C. (2020). p53 Integrates Temporal WDR5 Inputs during Neuroectoderm and Mesoderm Differentiation of Mouse Embryonic Stem Cells. Cell reports, 30(2), 465-480.e6.
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4

Histone Extraction and Immunoprecipitation from Embryonic Stem Cells

Cited 1 time
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ESCs or EBs were lysed with RIPA buffer (Pierce) in the presence of EDTA free protease inhibitor cocktail (Sigma). Histone extraction on EBs was performed using a histone extraction kit (Abcam) as described previously (Khan et al., 2015 (link)). Protein concentration was determined using Pierce BCA protein assay kit (ThermoFisher Scientific). 1.0 μg of histone extracts, or 10 μg of whole cell lysate was resolved on 4%–20% precast gel (Bio-Rad) was transferred to 0.45 μm PVDF membrane (Millipore). For immunoprecipitation, day 2 WT and Wdr5KO EBs were harvested and lysed in RIPA buffer with EDTA free protease inhibitor cocktail (Sigma). 1 mg sonicated cell lysates were subjected to immunoprecipitation using WDR5 antibody (Bethyl). The following primary antibodies were used for probing: anti-HA (1:10,000, Abcam, ab9110), anti-WDR5 (1:5000, R&D), anti-WDR5 (1:5,000, Bethyl), anti-Flag (1:2,000, Sigma), anti-H3 (1:10,000, Abcam), anti-H3K4Me1 (1:5,000, Millipore), anti-H3K4Me2 (1:10,000, Millipore), anti-H3K4Me3 (1:10,000, Abcam), anti-P53 (1:5,000, Cell Signaling), anti-Tubulin (1:10,000, Cell signaling), anti-β-Actin (1:10,000, Cell signaling), anti-Phospho-p53 (Ser15) Antibody (1:5,000, Cell Signaling), anti-Phospho-p53 (Ser392) Antibody (1:5,000, Abcam), anti-Acetyl-p53 (K305) Antibody (1:5,000, Abcam), anti-Actyl-p53 (K379) Antibody (1:5,000, Cell Signaling).
Li Q., Mao F., Zhou B., Huang Y., Zou Z., denDekker A.D., Xu J., Hou S., Liu J., Dou Y, & Rao R.C. (2020). p53 Integrates Temporal WDR5 Inputs during Neuroectoderm and Mesoderm Differentiation of Mouse Embryonic Stem Cells. Cell reports, 30(2), 465-480.e6.
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5

In Situ Proximity Ligation Assay of MuSCs

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The in situ proximity ligation assay (PLA) was performed on fixed primary proliferating MuSCs using the DuoLink PLA fluorescence technology (Sigma‐Aldrich#DUO92101), following the manufacturer′s protocol. About 2,000 isolated muscle satellite cells were seeded per well of a 96‐well plate and grown to a confluence of about 80%. Cells were fixed using 4% PFA for 7 min at room temperature. Myoblasts were then permeabilized with 0.3% Triton X‐100 in PBS three times for 5 min at room temperature. After two washing steps with PBS, cells were incubated with blocking solution in a humid chamber for 60 min at 37°C followed by incubation with primary antibodies for 1 h at room temperature. The assay was always performed with pairs of antibodies produced in mouse or rabbit, diluted 1:5,000 in antibody diluent. An anti‐V5 tag antibody (Thermo Scientific; 37–7,500) recognizing the endogenous V5‐tagged INO80 was used in combination with anti‐YY1 (Cell Signaling#46395), anti‐WDR5 (Bethyl#A302‐429A), and anti‐Ruvbl2 (Bethyl#A302‐536A) antibodies, respectively. PLA probe incubation, ligation, and signal amplification were performed according to the manufacturer's protocol. After two washing steps with PBS, DAPI was diluted 1:5,000 in PBS and added to the cells for 5 min at room temperature.
Schutt C., Hallmann A., Hachim S., Klockner I., Valussi M., Atzberger A., Graumann J., Braun T, & Boettger T. (2020). Linc‐MYH configures INO80 to regulate muscle stem cell numbers and skeletal muscle hypertrophy. The EMBO Journal, 39(22), e105098.
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6

Chromatin Immunoprecipitation Assay Protocol

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ChIP assays were performed essentially as described before[20 (link)–21 (link)]. Aliquots of lysates containing 200 μg of nuclear protein were used for each immunoprecipitation reaction with anti-MKL1 (Santa Cruz), anti-BRG1 (Abcam), anti-WDR5 (Bethyl Laboratories), anti-dimethylated H3K4 (Millipore/Upstate), and anti-trimethylated H3K4 (Millipore/Upstate). For re-ChIP, immune complexes were eluted with the elution buffer (1% SDS, 100mmol/L NaCO3), diluted with the re-ChIP buffer (1% Triton X-100, 2 mmol/L EDTA, 150 mmol/L NaCl, 20 mmol/L Tris pH 8.1), and subject to immunoprecipitation with a second antibody of interest. All experiments were repeated three times.
Xu W., Zhao Q., Wu M., Fang M, & Xu Y. (2017). MKL1 mediates TNF-α induced pro-inflammatory transcription by bridging the crosstalk between BRG1 and WDR5. Journal of Biomedical Research, 33(3), 164-172.
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7

Antibody Validation for Protein Interactions

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Antibodies are as follows: anti-HA (Abcam, ab9110), anti-AKAP95 (Santa Cruz Biotechnology, sc-10766), anti-His (Santa Cruz Biotechnology, sc-803), anti-GAPDH (Chemicon, MAB374), anti-FLAG (Sigma, A8592, for blotting), anti-FLAG [M2 beads] (Sigma, A2220), and anti-H3K4me3 (Millipore, 07–473), anti-RBBP5 (Bethyl Laboratories, A300–109A), anti-ASH2L (Bethyl Laboratories, A300–107A), anti-WDR5 (Bethyl Laboratories, A302–430A).
Shah K.K., Whitaker R.H., Busby T., Hu J., Shi B., Wang Z., Zang C., Placzek W.J, & Jiang H. (2019). Specific inhibition of DPY30 activity by ASH2L-derived peptides suppresses blood cancer cell growth. Experimental cell research, 382(2), 111485.
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8

Protein Interaction and Transcriptional Regulation Assays

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All plasmids about BAP18 were introduced in our previous work (39 (link)). ERα expression plasmid was cloned into pSG5 plasmid and 3 estrogen response elements were cloned made into pGL-ERE-adML reporter plasmid to perform luciferase reporter assay.
The antibodies were used in our study as follow: anti-BAP18 (Bethyl #A304-207A-1), anti-ERα (Cell signaling #D8H8), anti-MYC (Thermo Fisher #9E10), anti-CyclinD1 (Cell signaling #DCS6), anti-GAPDH (Kangchen #KC5G4), anti-WDR5 (Bethyl #A302-429A-2), anti-ASH2L (Bethyl #A300-107A-2), anti-DPY30 (Abcam #ab187690), anti-H3K4me3/H3ac/H3K9ac/H4ac/H4K16ac (Millipore), anti-Rabbit/Mouse (ABclonal), anti-IgG (Proteintech#10238-1-AP), anti-GFP (Sigma #G1544) and anti-FLAG (Proteintech#20543-1-AP).
Sun G., Wang C., Wang S., Sun H., Zeng K., Zou R., Lin L., Liu W., Sun N., Song H., Liu W., Zhou T., Jin F., Shan Z, & Zhao Y. (2020). An H3K4me3 reader, BAP18 as an adaptor of COMPASS-like core subunits co-activates ERα action and associates with the sensitivity of antiestrogen in breast cancer. Nucleic Acids Research, 48(19), 10768-10784.
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9

Spindle Extraction and Immunoblotting

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The samples were electrophoresed on a SDS-PAGE gel, transferred on nitrocellulose membrane (Amersham) and immunoblotted with anti-MLL C (1:250, Bethyl), anti-WDR5 (1:1000,Bethyl), anti-RbBP5 (1:1000, Bethyl), anti-Kif2A (1:1000, Abcam), anti-GFP (1:1000, Invitrogen), anti-GAPDH (1:10000, Sigma), anti-GST(1:10000, Abcam), anti-H3S10P (abcam), and anti-MLL2 (1:250, Bethyl).
Proteins were detected with Licor-Biosciences imaging system as described previously (Tyagi et al., 2007) using IR Dye 800CW conjugated anti-mouse IgG( Licor Biosciences), IR Dye 680 LT conjugated anti-rabbit IgG ( Licor Biosciences).
Spindle Extraction U2OS cells were seeded in 150 x 25mm tissue culture plates. At 60-70% confluency cells were synchronized by thymidine-nocodazole block as described (Harper, 2005) . The cells were then harvested by mitotic shake off method. The spindle extraction was done as described (Sillje ´and Nigg, 2006) . The spindle extract was subjected to SDS-PAGE followed by immunoblotting.
Ali A., Veeranki S.N., Chinchole A, & Tyagi S. (2017). MLL/WDR5 Complex Regulates Kif2A Localization to Ensure Chromosome Congression and Proper Spindle Assembly during Mitosis. Developmental cell, 41(6).
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