Tissue was dissected in cold 1xPBS and fixed in 4%PFA for 2 h. Following washes in 1xPBS, samples were blocked using a solution containing 5% normal goat serum in PBSTx for 1 h. The blocking solution was replaced with a primary antibody solution containing 1:100 DNMT1 antibody (Abcam ab188453), and incubated overnight at room temperature. Negative controls without primary antibody were incubated overnight in the blocking solution at room temperature in parallel. All samples were washed and incubated in the secondary solution including 1:500 secondary antibody (Donkey anti-rabbit, AlexaFluor 594, Invitrogen A21207) and 1:500 DAPI for 2 h prior to mounting in DAKO fluorescence mounting medium. Images were captured and processed as described above.
Dnmt1 antibody
Manufactured by Abcam
Sourced in United Kingdom
DNMT1 antibody is a protein that recognizes and binds to the DNA methyltransferase 1 enzyme. This enzyme is responsible for maintaining DNA methylation patterns during cell division.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Protein a g magnetic beads, by Selleck Chemicals (1 mentions)
Akt antibody, by Cell Signaling Technology (1 mentions)
Hydralazine hydrochloride, by Merck Group (1 mentions)
Procainamide, by Merck Group (1 mentions)
Dihydroethidium, by Merck Group (1 mentions)
Q vd oph, by R&D Systems (1 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Lsm 710 nlo confocal microscope, by Zeiss (1 mentions)
γh2ax antibody, by Merck Group (1 mentions)
Ez magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Decitabine, by Selleck Chemicals (1 mentions)
Parp 1 antibody, by Abcam (1 mentions)
Gemcitabine, by Selleck Chemicals (1 mentions)
Trametinib, by Selleck Chemicals (1 mentions)
P005091, by Selleck Chemicals (1 mentions)
Gapdh antibody, by Abcam (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
13 protocols using dnmt1 antibody
1
DNMT1 Immunohistochemistry in Tissue Samples
2
Investigating Pp6 Interactions with Dnmt1 and Akt
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To determine the interaction between Pp6 and Dnmt1 or Akt, EL-4 cells were lysed by RIPA buffer or WB-IP buffer (Beyotime). Equal amounts of lysates were incubated with protein A/G magnetic beads (Bimake) and antibodies, including Pp6 antibody (Millipore), Dnmt1 antibody (Abcam), or Akt antibody (Cell Signaling Technology). The prepared mixture was incubated overnight at 4 °C with agitation. The mixture was washed three times with PBS or lysis buffer and boiled in sample buffer. Then, the prepared immunoprecipitation complex was eluted for Western blot analysis to determine the interacting protein levels. Total cell protein was used as an input. The specificity of antibodies used for immunoprecipitation was routinely validated by using a negative control IgG.
3
Apoptosis Pathway Regulation Analysis
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Hydralazine hydrochloride, procainamide, dihydroethidium (DHE) and mouse anti-β-actin monoclonal antibody were from Sigma-Aldrich (ST. Louis, MO). The fluorescent cationic lipophilic dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6 (3)) and Alexa Fluor 488-labeled goat anti-mouse were obtained from Molecular Probes (Carlsbad, CA). Manganese-porphyrin Mn(III)TMPyP was from Cayman Chemical (Ann Arbor, MI). Q-VD-OPh, a wide-spectrum caspase inhibitor, and anti-human caspase-9 monoclonal antibody were from R&D Systems (Minneapolis, MN). Cytofix/cytoperm was from BD Biosciences (San José, CA). Anti-Bak (Ab-1) monoclonal antibody and anti-Chk1 (pSer317) rabbit polyclonal antibody were obtained from Calbiochem (Darmstadt, Gemany). Anti-phospho-histone H2AX (Ser139) monoclonal antibody was from Upstate/Millipore (Billerica, MA). Anti-human caspase-8 monoclonal antibody was purchased from Cell Diagnostica (Munster, Germany). Anti-human caspase-3 polyclonal antibody was obtained from Stressgen (Ann Arbor, MI). Mouse monoclonal DNMT1 antibody was from Abcam (Cambridge, UK).
4
Protein Extraction and Western Blot Analysis
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Total protein of the whole embryos was extracted by RIPA lysis buffer (YOBIBIO, Shanghai, UBI1003). The protein concentrations were identified using A BCA protein kit from Thermo Fisher (Rockford, IL). Following separation on SDS‐polyacrylamide gels, proteins were transferred onto appropriate PVDF membranes (Immobilon‐P; Millipore, Bedford, MA, USA) following the manufacture's inductions. The membranes were blocked for 1 hour with 5% non‐fat dried milk in TBST at room temperature (RT), and then incubated overnight with Dnmt1 antibody (1:500 dilution, Abcam, UK, ab13537) or anti‐GAPDH antibody (1:1000 dilution, GNI, Japan, 4310‐GH) at 4 °C. The ECL kit (Pierce) was used to visualize the immunoreactive bands and the intensities of the bands were quantified with Fiji (National Institutes of Health).
5
In Situ DNMT1 and γH2AX Detection
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For detection of DNMT1 and γH2AX in situ following drug treatment, cells were suspended in PBS and added to the EZ cytofunnel chamber to prepare cytospins. They were centrifuged at 600 rpm for 10 min, with approximately 200,000 cells per slide, which were then fixed in 4% paraformaldehyde for 15 min at room temperature; followed by permeabilizing with 0.1% Triton-X100 PBS solution at RT for 10 min. For staining, γH2AX antibody in 1:300 dilution (Millipore Sigma) and DNMT1 antibody in 1:500 dilution (Abcam) was incubated overnight at 4 °C. Antibody attachment was reported using a conjugate reporter stain of either anti-mouse Alexa Fluor 555 or anti-rabbit Alexa Fluor 488. Image capture was conducted on a Carl Zeiss LSM 710 NLO confocal microscope. The areas of cells were selected based on DAPI staining, excluding thick cellular areas with overlapping cells, areas with artifacts, or poor staining. Average fluorescence intensity was calculated using ZEN 2009 Image Pro Premier (ZEISS).
6
Formaldehyde-based DNMT1 ChIP-qPCR
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A549 cells were treated with formaldehyde and incubated for 10 minutes, followed by sonication to achieve the majority of DNA fragments with 200 to 300 bp. The fragmented chromatin were immunoprecipitated with DNMT1 antibody (Abcam) or IgG as control. Precipitated chromatin DNA was recovered and measured by qPCR.
7
RIP Assay of DNMT1 Binding
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RIP assay was performed by using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) according to the manufacturer’s instructions. The DNMT1 antibody was used for RIP (Abcam, Cambridge, UK). The co-precipitated RNAs were detected by reverse transcription PCR and real-time PCR. Total RNAs (input controls) and IgG were assayed simultaneously to establish that the detected signals were the result of RNAs specifically binding to DNMT1. The optical density was scanned by Image J and normalized to input group.
8
FBP1 Regulation in Cancer Metabolism
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Gemcitabine (#S1714), MK2206 (#S1078), Trametinib (#S2673), Olaparib (#S1060), P005091 (#S7132), Decitabine (# S1200) were purchased from Selleck Chemicals (Shanghai, China). FBP1 antibody (# ab109732, working dilution 1 : 1000), GAPDH antibody (# ab8245, working dilution 1 : 5000), DNMT1 antibody (# ab92314, working dilution 1 : 1000), and PARP1 antibody (# ab32138, working dilution 1 : 1000) were purchased from Abcam. USP7 antibody (# 66514‐1‐Ig, working dilution 1 : 1000) was obtained from Proteintech (Wuhan, China). The KOD‐plus‐mutagenesis kit (#SMK101, TOYOBO LIFE SCIENCE, Osaka, Japan) was used to generate FBP1 mutants mentioned in the results.
9
Ubiquitination of DNMT1 in COS-7 cells
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COS-7 cells were co-transfected with a mixture of GFP or GFP-SET7, 3xFLAG or 3xFLAG-PHF20L1, HA-ubiquitin as well as DsRed-DNMT1 plasmids, and Transpass D2 transfection reagent (New England Biolabs) at a ratio of 1:3 μg/μl for 24 h. Transfected COS-7 cells were treated with 10 μm MG132 for 12 h and lysed as described previously (27 (link)). Cell lysates (100–200 μg) were then immunoprecipitated with DNMT1 antibody (Abcam, catalog no. ab92453). Ubiquitination was detected as described previously (27 (link)).
10
RPTECs-based in vitro DN Model
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RPTECs were cultured on the EZ slides (Merck Millipore). Treatment of Ang-II, AGE and HG was given to the cells and subsequent transfection with miR29b mimics and miR29b inhibitors was carried out. The cells were fixed using 4% paraformaldehyde. RPTECs based in vitro DN model was incubated with primary DNMT3A antibody (1:1000, Abcam), DNMT3B antibody (4 μg/ml, Sigma), DNMT1 antibody (1 μg/ml, Abcam) for overnight at 4 ºC. After overnight incubation with primary antibody, the slides were washed with PBS (3 times). The slides were then incubated with secondary antibody goat anti-rabbit IgG-FITC for 2 h. After complete incubation, slides were again washed with PBS (3 times) and counter stained with DAPI for cell nuclei. Slides were then observed under the microscope and images were quantified using Image J software.
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