Frozen or paraffin-embedded skin specimens were sectioned to consecutive levels of thickness. Sections were stained with hematoxylin and eosin (H&E) according to standard procedures. For immunostaining analyses of tissues, the sections were deparaffinized and stained with antibodies against G9A (cat. ab185050, Abcam, 1:500), Ki67 (cat. ab16667, Abcam, 1:200), PCNA (cat. ab15497, Abcam, 1:200).
Ab185050
Manufactured by Abcam
Sourced in United States
Ab185050 is a lab equipment product offered by Abcam. It is a device designed for scientific research purposes. The core function of this product is to facilitate specific laboratory procedures or analyses.
Automatically generated - may contain errors
Lab products found in correlation
Ab1220, by Abcam (6 mentions)
Ab181602, by Abcam (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Ab86934, by Abcam (2 mentions)
Celena s digital imaging system, by Logos Biosystems (2 mentions)
Ab1791, by Abcam (2 mentions)
Ab9045, by Abcam (2 mentions)
Ab6002, by Abcam (2 mentions)
Ab191389, by Abcam (2 mentions)
Anti e4bp4 antibody sc 74415, by Santa Cruz Biotechnology (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Antibeta actin ab8226, by Abcam (2 mentions)
Anti kdm4c nbp1 49600, by Novus Biologicals (2 mentions)
Ab15497, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ecl kit, by Beyotime (1 mentions)
19 protocols using ab185050
1
Histological Analysis of Skin Samples
2
Proteomic Analysis of Myocardial Tissues
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Myocardial tissues and cells were collected and lysed using ultrasonic cell disruption on ice in radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) for 30 min. The samples were centrifuged at 12,000 × g for 30 min at 4 °C to collect the supernatant. Protein concentrations were determined using a BCA protein assay kit (Sigma-Aldrich Chemical Company, St Louis, MO, USA). Protein lysates (30 μg) were loaded onto 12% sodium dodecyl sulfate–polyacrylamide gels for electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). One hour of blocking was done at room temperature with 5% BSA (Gibco, Carlsbad, CA, USA). Membranes were incubated at 4 °C overnight with primary antibodies as follows: G9a (1:1000, ab185050, Abcam, Cambridge, UK), BDNF (1:2000, GTX132621, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), H3K9me2 (1:1000, GTX54102, GeneTex), α-SMA (1:2000, GTX100034, GeneTex), FN1 (1:1000, ab268020, Abcam), p-TrkB (1:1000, ab229908, Abcam), TrkB (1:1000, GTX54857, GeneTex), GAPDH (1:10,000, ab181602, Abcam). After a wash, blots were incubated with goat anti-rabbit IgG-horseradish peroxidase (1:5000, ab6721, Abcam) for 60 min at room temperature. The blot was visualized with an ECL kit (Beyotime) and quantified with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
3
ChIP Profiling of H3K9me2 and G9a in Myoblasts
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ChIP experiments on MuSC-derived myoblasts were performed on chromatin extracts according to the manufacturer's protocol (MAGnify ChIP; Life Technologies) by O.N. incubation with 3 μg of immobilized anti-H3K9me2 (Abcam ab1220; RRID:AB_449854 ) or rabbit IgG (14-4616-82; RRID:AB_2865072 ) antibodies. A standard curve was generated for each primer pair testing 5-point dilutions of input sample. Fold enrichment was quantified using qRT-PCR (SYBR Green; Qiagen) and calculated as a percentage of input chromatin (% Inp). Data from GAP-SCR vs GAP‐REW conditions represent the mean of six independent experiments ± SEM. For G9a ChIP experiments on proliferating C2C12, crosslinking was performed by adding DSG (di-succinimidyl glutarate; Santa Cruz) at a final concentration of 2 mM for 45 min at RT. Then, formaldehyde (Sigma) was added to culture medium to a final concentration of 1% for 10 min at RT and stopped by glycine to a final concentration of 0.125 M. Chromatin was extracted as described in Mozzetta et al., 2014 (link) and immunoprecipitated with 5 μg of G9a (Abcam, ab185050; RRID:AB_2792982 ) or rabbit IgG (14-4616-82; RRID:AB_2865072 ) antibodies carried out overnight at 4°C. Sequences of the oligonucleotides used for ChIP analyses are reported in Supplementary file 1 .
4
Immunofluorescence Assay for EHMT2 and TNFAIP1
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Cultured cells were fixed in 4% paraformaldehyde at room temperature for 10 min, permeabilized in 0.5% Triton X-100 (Sigma-Aldrich) in PBS for 10 min, and blocked with 5% bovine serum albumin in PBS for 30 min. Fixed cells were incubated with anti-EHMT2 antibody (ab185050; Abcam) and anti-TNFAIP1 antibody (ab86934; Abcam) overnight at 4 °C and stained with Alexa Fluor-conjugated secondary antibodies (Life Technologies). Fluorescent images were obtained using a CELENA S Digital Imaging System (Logos Biosystems).
5
Protein Expression Analysis Protocol
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Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA). After separation by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred onto polyvinylidene difluoride membranes (PVDF, Millipore, Burlington, Massachusetts, USA) for 120 min Then, the cells were blocked with TBST containing 5% nonfat skimmed milk and incubated with primary antibodies (anti-CCDC8, ab235780, 1:2000, Abcam; anti-GAPDH, ab9485, 1:5000; Abcam; anti-H3, ab1791, 1:8000, Abcam; anti-H3K9me3, ab8898, 1:5000, Abcam; anti-G9a, ab185050, 1:2000, Abcam). Subsequently, secondary antibodies were added to culture the cells (goat anti-rabbit IgG H&L, ab6721, 1:10,000; goat anti-mouse IgG H&L, ab6789, 1:2000; Abcam). The signal detection was conducted by means of an ECL system (Life Technology, USA). GAPDH and H3 was regarded as an internal reference.
6
Western Blot Analysis of Histone Modifications
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Cells in each group were lysed in radio-immunoprecipitation assay buffer containing protease inhibitors (Sigma-Aldrich). The lysate was centrifuged at 16000 g and 4°C for 20 min to collect the supernatants. The concentration of protein extracted from cells was tested using the Pierce bicinchoninic acid assay kit (Beyotime, Shanghai, China). Then, the protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, United States). The membranes were blocked with 5% skim milk for 2 h and cultured with the primary antibodies at 4°C overnight. Thereafter, the membranes were cultured with the secondary antibody for 1 h, and developed and visualized using the enhanced chemiluminescence reagent. The gray value of the target band was analyzed by Image J software (National Institutes of Health, MD, United States). The antibodies used were as follows: H3K27me3 (1:1000, ab6002, Abcam), β-actin (1:1000, ab8227, Abcam), H3K9me1 (1:1000, ab9045, Abcam), H3K9me2 (1:1000, ab1220, Abcam), EZH1 (1:1000, ab189833, Abcam), EZH2 (1: 1000, ab150433, Abcam), and EHMT2 (1:1000, ab185050, Abcam).
7
ChIP Assay of BV-2 Cells
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In accordance with the instructions of an EZ ChIP kit (Millipore, Germany), transfected BV-2 cells were fixed with 1% formaldehyde to crosslink DNA–protein complexes for 10 min. After that, the crosslinking was terminated by adding glycine for 5 min. The cells (106) were lysed with 100 µL SDS lysis buffer (SDS lysis buffer + proteinase inhibitor cocktail II). Next, the DNA from the cell lysate was ultrasonically broken into 200–1,000 bp fragments. IgG antibody (Millipore), EHMT2 monoclonal antibody (ab185050; Abcam), and H3K4Me3 monoclonal antibody (ab213224; Abcam) were used for ChIP assay. The purified DNA products obtained were detected by qRT-PCR to confirm whether the DNA precipitated by antibodies contained the DNA sequence of the target gene and its relative content.
8
Immunofluorescence and Immunohistochemistry of Tumor Sections
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Immunofluorescence of tumor sections was performed as previously described2 (link), with antibodies for pro-SPC (1:200, Santa Cruz), G9a (ab185050, Abcam, 1:200), Notch3 (5276S, Cell Signaling, 1:100), Mmp10 (ab59437, Abcam, 1:100), Mmp13, (ab39012, Abcam, 1:100), and Mcpt4 (ab92368, Abcam, 1:100). Immunohistochemistry was performed as described2 (link) with anti-Ki-67 (1:10,000, Novocastra) antibodies and developed using Vectastain Elite ABC Kit (Vector Labs). Imaging was performed with a Nikon 90i camera and NIS-Elements software and processed with NIS-Elements and Adobe Photoshop.
9
Chromatin Immunoprecipitation Protocol
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In accordance with the manufacturer's instructions, ChIP assay was performed by using a ChIP assay kit (Upstate/Millipore, Billerica, MA). Briefly, the indicated HEY cells were grown to 70%–80% confluence on a 100 mm culture dish and treated with 1% formaldehyde for 10 min, which were collected and sonicated to shear the DNA into small, uniform fragments. Equal aliquots of chromatin supernatants were immuno-precipitated overnight at 4°C using ZNF711 (ab254776, Abcam), or anti-JHDM2A (ab191389, Abcam), anti-Flag (F3165, Sigma), anti-Pol II (Millipore, 07-1802), anti-EHMT2 (ab185050, Abcam), anti-H3K9me2 (ab1220, Abcam), anti- H3K9me1 (ab176880, Abcam), anti-H3K27me3 (ab6002, Abcam), anti-H4R17me1 (PTM-697, PTM biolabs, Chicago, IL, USA), antibodies, or an anti-anti-IgG antibody (a negative control, Millipore, Billerica, MA) respectively, with protein G magnetic beads (10003D, Invitrogen). A total of 2 μg of each antibody was used per 107 cells for each ChIP assay. The cross-linked protein/DNA complexes were collected by magnetic pull down, and subsequently eluted from beads using elution buffer. After reverse cross-linking the protein/DNA complexes to free DNA, the enriched DNA fragments was conducted by PCR assays by specific primers. All ChIP primers used in this study are listed in Supplementary Table S3.
10
Immunoblotting analysis of epigenetic and apoptotic markers
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Floating and attached cells were lysed in Radioimmunoprecipitation assay (RIPA) buffer. Protein concentration was determined by using Micro BCA TM protein assay kit (Thermo Fisher). Immunoblotting was performed as described previously (5 (link)). The following antibodies were used to detect G9a (ab185050, Abcam, RRID:AB_2792982 ), cPARP (ab32064, Abcam, RRID:AB_777102 ), MYCN (B8.48, Santa Cruz, SC-53993, RRID:AB_831602 ), cCaspase 3 (9664, Cell Signaling Technology, RRID:AB_2070042 ), LC3B (L7543, Sigma, RRID:AB_796155 ), histone H3 (ab10799, Abcam, RRID:AB_470239 ), dimethyl K9 histone H3 (ab1220, Abcam, RRID:AB_449854 ), and β-Actin (A3854, Sigma, RRID:AB_262011 ), according to manufacturer's instructions.
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