SGC0946 and EPZ004777 were purchased from Selleck Chemicals (TX, USA). Antibodies used in ChIP were as follows: anti-H3K4me3 (ab8580; Abcam, CA, USA), anti-H3K9me3 (ab8898; Abcam), anti-H3K27me3 (07-449; Millipore, MA, USA), anti-H3K36me3 (ab9050; Abcam), anti-H3K79me2/me3 (ab2621; Abcam), anti-H4K20me3 (ab9053; Abcam), anti-C/EBPβ (ab18336; Abcam), and anti-DOT1L (ab72454; Abcam). Primary antibodies used in western blot and IHC were as follows: anti-C/EBPβ (SAB4500112; Sigma, St. Louis, MO, USA), anti-DOT1L (ab72454; Abcam), anti-RICTOR (GTX104617; GeneTex, USA), anti-NEIL3 (11621-1-AP; Proteintech, Wuhan, China), anti-NIPBL (18792-1-AP; Proteintech), anti-PHF20 (ab157192; Abcam), anti-SLC38A1 (12039-1-AP; Proteintech), anti-AMOTL1 (ab171976; Abcam), anti-pEGFR (Tyr1148; cat. no. #4404; Cell Signaling Technology, Danvers, MA, USA), anti-MEK1/2 (Ser217/221; cat. no. #9154; Cell Signaling Technology), anti-pERK1/2 (Thr202/Tyr204; cat. no. #4370; Cell Signaling Technology), anti-pPI3K p85 (Tyr458)/p55 (Tyr199; cat. no. #4228; Cell Signaling Technology), anti-pAKT (Thr308; cat. no. #13038; Cell Signaling Technology), anti-pSTAT1 (Tyr701; cat. no. #7649; Cell Signaling Technology), anti-pSTAT3 (Tyr705; cat. no. #9145; Cell Signaling Technology), and anti-GAPDH (10494-1-AP).
Ab72454
Manufactured by Abcam
Sourced in United States
Ab72454 is a laboratory equipment product offered by Abcam. It is a device designed for a specific core function in the lab environment. No further details can be provided in a concise, unbiased, and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab3594, by Abcam (3 mentions)
Ab2621, by Abcam (3 mentions)
Ab2886, by Abcam (2 mentions)
Sgc0946, by Selleck Chemicals (2 mentions)
Epz004777, by Selleck Chemicals (2 mentions)
Ab8898, by Abcam (2 mentions)
Ab9050, by Abcam (2 mentions)
Anti β actin a1978, by Merck Group (1 mentions)
Anti ercc1, by Cell Signaling Technology (1 mentions)
Anti p53 7f5, by Cell Signaling Technology (1 mentions)
Anti ddb1 d4c8, by Cell Signaling Technology (1 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (1 mentions)
Ab22656, by Abcam (1 mentions)
Ab254530, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Ab7766, by Abcam (1 mentions)
Ab32063, by Abcam (1 mentions)
Ab24684, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
8 protocols using ab72454
1
Chromatin Immunoprecipitation Antibodies and Reagents
2
SDS-PAGE and Western Blotting Analysis
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SDS-PAGE and Western blotting analyses were performed using standard protocols. For protein detection, the following primary antibodies according to their manufacturer’s instructions were used: mouse monoclonal anti-β-actin (A1978, Sigma-Aldrich, St Louis, MO); rabbit polyclonal anti-menin (A300-105A, Thermo Fisher Scientific) and anti-KMT4/Dot1L (ab72454, Abcam, Cambridge, UK).
3
Comprehensive Western Blotting Antibody Panel
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The following antibodies used in Western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA): anti-HA-tag (6E2) (1:1000, #2367, Cell Signaling Technology), anti-p53 (7F5) (1:1000, #2527, Cell Signaling Technology), anti-DDB-1 (D4C8) (1:1000, #6998, Cell Signaling Technology), anti-DDB-2 (D4C4) (1:1000, #5416, Cell Signaling Technology) anti-ERCC1 (1:1000, #3885, Cell Signaling Technology), and anti-XPC (D1M5Y) (1:1000, #14768, Cell Signaling Technology). The Polyclonal anti-KMT4/DOT1L (1:2000, ab72454, Abcam) antibody, anti-XPA (1:2000, ab85914, Abcam), polyclonal di-methylated H3K79 antibody (1:1000, ab3594, Abcam), polyclonal to mono-methylated H3K79 antibody (1:500, ab2886, Abcam), and polyclonal tri-methylated H3K79 antibody (1:1000, ab2621, Abcam) were purchased from Abcam. TFIIH p62 Antibody (1:500, Q-19) was purchased from Santa Cruz Biotechnologies, Inc. Other antibodies include monoclonal Anti-β-Actin-Peroxidase antibody (1:25000, AC-15, Sigma Aldrich), monoclonal ANTI-FLAG® M2 antibody (1:1000, Sigma Aldrich), and anti-H3 (1:500, 865R2, Thermo Fisher Scientific Inc.). Most important uncropped blot images are shown in Supplementary Fig. 11 .
4
Immunofluorescence Analysis of Cell Surface Markers
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Prior to being embedded in paraffin, human tissues have been preserved in 10% neutral-buffered formalin. Utilising Tris/EDTA antigen retrieval buffer, sections had been exposed to antigen retrieval for 20 minutes during 95°C. For IF, sections have been subjected to primary antibodies against CD44 (Abcam, mouse# ab254530; 1:200 dilution), DOT1L (Abcam, rabbit# ab72454; 1:100 dilution) and CTNNB1 (Abcam, mouse#ab22656; 1:100 dilution) during 4°C overnight, after which the goat anti-mouse IgG HL (Alexa Fluor 488 1:100 dilution) as well as goat anti-IgG HL (Alexa Fluor 594 1:100 dilution) were incubated with fluorophore-conjugated secondary antibodies. For thirty minutes, DAPI (Beyotime, P0131) was utilised during room temperature. The sections have been examined using an Olympus fluorescence microscope.
5
Investigating Epigenetic Regulation by DOT1L
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The following antibodies were used for immunoprecipitation and Western blot analyses: C-terminal anti-ERα (F-10 sc-8002, Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-estrogen Receptor Alpha (ab32063, Abcam, Cambridge, UK), rabbit polyclonal anti-DOT1L (A300-953A, Bethyl Laboratories, Montgomery, Alabama), β-actin (A1978, Sigma Aldrich, Milan, Italy), Rabbit anti-KMT4/DOT1L (ab72454), anti-Histone H3, total, (ab1791), anti-H3K79me1 (ab2886), anti-H3k79me2 (ab3594), anti-H3K79me3 (ab2621), anti-H3K4me3 (ab7766), anti-H3K27me3 (ab24684) from Abcam, anti-Rabbit IgG Isotype Control (31235, Thermo-Fisher), and the anti-Mouse IgG antibody (RM104, Aurogene, Rome, Italy).
Cells were treated with the following compounds: DOT1L inhibitors EPZ004777 (S7353), Pinometostat (EPZ5676)(S7062), SGC0946 (S7079), all from Selleckchem, and with 4-hydroxytamoxifen (4-OHT) (H7904, Sigma-Aldrich), Fulvestrant (ICI 182,780) (I4409, Sigma-Aldrich), and β-estradiol (E887-5G, Sigma-Aldrich) or vehicles (DMSO and/or EtOH), according to different experimental settings.
Cells were treated with the following compounds: DOT1L inhibitors EPZ004777 (S7353), Pinometostat (EPZ5676)(S7062), SGC0946 (S7079), all from Selleckchem, and with 4-hydroxytamoxifen (4-OHT) (H7904, Sigma-Aldrich), Fulvestrant (ICI 182,780) (I4409, Sigma-Aldrich), and β-estradiol (E887-5G, Sigma-Aldrich) or vehicles (DMSO and/or EtOH), according to different experimental settings.
6
Immunoprecipitation and Chromatin Fractionation
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Briefly, cells were washed twice with ice-cold PBS and lysed in lysis buffer containing 50 mM Tris pH 7.4; 1% Triton X-100; 0.5 mM EDTA; 0.5 mM EGTA; 150 mM NaCl; 10% Glycerol; 1 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor cocktail (Roche) on ice for 30 min. The supernatant was collected after centrifugation at 15,000 × g for 15 min at 4 °C, and 500 µg of total cell lysate was treated by DNase (15 U mL−1, Pierce), precleared by 20 µL Protein G Agarose Beads (Thermo Fisher Scientific Inc.), and then incubated with primary antibody, including anti-DOT1L (ab72454, Abcam) and anti-XPC (#14768, Cell signaling) overnight at 4 °C. In all, 20 µl of Protein A/G Agarose Beads was added into the samples with rotation at 4 °C for 1 h. After three washes with 1 mL of lysis buffer, the bound proteins were released by boiling in 30 μL of sodium dodecyl sulfate loading buffer and detected as described above. The chromatin fractionation was extracted with the Chromatin Extraction Kit (ab117152, Abcam) from the cells and assayed with western blot.
7
Comprehensive Protein Expression Analysis
8
Immunoblotting analysis of Dot1L, NUPR1, GAPDH, and pAKT
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Cells were lysed in loading buffer. The Lowry protein assay was used to calculate protein concentrations. Then we used SDS-PAGE to separate 10 μg of protein lysate, which was then transferred to a PVDF membrane (Solarbio, Beijing, China). The indicated proteins were detected by immunoblotting with specific antibodies in 5% bovine serum albumin. The antibodies used were as follows: anti-Dot1L (ab72454, Abcam, Cambridge, UK), anti-NUPR1 (ab161980, Abcam), anti-GAPDH (sc-20357, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-pAKT (4506, Cell Signaling Technology, Danvers, MA, USA).
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