The following primary antibodies were used: anti-AGEs (1:2000, 6D12, Trans Genic Inc., Japan), H3K4me1 (1:10000, ab176877, Abcam, United States), H3K4me2 (1:10000, ab32356, Abcam, United States), H3K4me3 (1:5000, ab213224, Abcam, United States), histone H3 (1:10000, ab176842, Abcam, United States), LSD1 (1:2500, 2184, Cell Signaling Technology, United States), PHF8 (1:5000, ab280887, Abcam, United States), Bax (1:5000, 50599-2-Ig, Proteintech, China), Bcl-2 (1:4000, A0208, Abclonal, China), and β-actin (1:2000, K101527P, Solarbio, China).
Ab176842
Manufactured by Abcam
Sourced in United States, China
Ab176842 is a lab equipment product offered by Abcam. It serves as a core functional component in laboratory settings, but a detailed description cannot be provided while maintaining an unbiased and purely factual approach. Additional information about the intended use or interpretation of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab8227, by Abcam (4 mentions)
Ab176877, by Abcam (3 mentions)
Ab32356, by Abcam (2 mentions)
Ab1162, by Abcam (2 mentions)
Ab176916, by Abcam (2 mentions)
Ab8580, by Abcam (2 mentions)
Ab200342, by Abcam (2 mentions)
Ab32158, by Abcam (2 mentions)
Ab186733, by Abcam (2 mentions)
Ab32072, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab213224, by Abcam (1 mentions)
A0208, by ABclonal (1 mentions)
Ab191698, by Abcam (1 mentions)
Ab94580, by Abcam (1 mentions)
Ab139311, by Abcam (1 mentions)
Ab131021, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Blocking buffer, by Beyotime (1 mentions)
14 protocols using ab176842
1
Comprehensive Epigenetic Protein Analysis
2
Immunoblotting Analysis of TET Proteins
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After treatments, NPC cells were washed with PBS and then lysed with cell lysis buffer (CST, USA). Whole cell lysates were centrifuged at 14,000g for 5 min at 4 °C to remove cell debris, and the supernatants were subjected to SDS-PAGE and immunoblotting, as previously described [25 ]. The following antibodies used in this study were purchased from Abcam (USA): rabbit anti-TET1 (ab191698), rabbit anti-TET2 (ab94580), rabbit anti-TET3 (ab139311), mouse anti-GAPDH (ab8245), rabbit anti-flag (ab1162), rabbit anti-PKM antibody (ab131021), and rabbit anti-Histone H3 antibody (ab176842).
3
Western Blot Analysis of Histone H3
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Total protein from ICC tumor and paracancerous specimens from 10 ICC patients were extracted and separated in 10% SDS-PAGE and then electro-transferred onto polyvinylidene difluoride membranes. After blocking in blocking buffer (Beyotime, China) for 1 h, incubated membranes with primary antibodies (Histone H3 (tri methyl K9), Abcam, ab176916; HistoneH3antibody, Abcam, ab176842;) and secondary antibodies (Goat anti-Rabbit, Proteintech, SA00001-2) were used. Performed the detection of target proteins using enhanced chemiluminescence (ECL) after washing with TBST. All experiments were performed in triplicate.
4
Western Blot Analysis of Mitochondrial Proteins
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Laemmli buffer containing 4% SDS, 10% β-mercaptoethanol (M6250; Sigma-Aldrich), 20% glycerol (GI345; Melford), 0.004% blue bromophenol (A2331,0025; AppliChem), and 0.125 M Tris-HCl was added to protein lysates, followed by boiling at 95°C for 10 min. Samples were separated on SDS-PAGE and transferred to a nitrocellulose (Macherey-Nagel) or a polyvinylidene difluoride (Merck) membrane. The membranes were blocked with 5% milk (A0830; PanReac AppliChem) and probed with the following antibodies: anti–β-Actin (ab8227; Abcam), anti–β-Tubulin (ab15568; Abcam), anti-CBS (14787-1-AP; Proteintech), anti-CSE (12217-1-AP; Proteintech), anti-MPST (HPA001240; Atlas Antibodies), anti-MTCO1 (ab14705; Abcam), anti-Citrate Synthase (ab96600; Abcam), anti-SOD2 (13141; Cell Signaling), anti-TOM40L (ab236421; Abcam), anti-TIM50 (ab109436; Abcam), anti-HIF1α (sc-13515; Santa Cruz Biotechnology), anti-H3 (ab176842; Abcam), anti-V5 (Merck), anti-HA (H6908; Sigma-Aldrich), and anti-Biotin (HRP conjugate; 5571; Cell Signaling). Immunoblots were next incubated with a secondary antibody (anti-rabbit [AP132P; Merck] or anti-mouse [7076; Cell Signaling]) and visualized using the Western HRP substrate (Merck). ImageJ software was used to quantify the expression levels of the proteins.
5
Protein Extraction and Western Blot Analysis
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Protein was extracted using lysis buffer and quantified by Pierce™ BCA Protein Assay Kit (Thermo, 23 227). 1 μL PMSF (0.1 mol/L) and 10 μL phosphatase inhibitor (10 g/L) were added per 1 mL lysis buffer immediately before use. Denatured proteins were resolved using 10%–12% SDS‐PAGE gels and blotted on PVDF membranes. Following blocking in 5% skim milk/0.2% Tween/dH2O, membranes were incubated with primary antibodies at 4°C for overnight. The primary antibodies used were anti‐ZSCAN4 (Abcam, ab106646; dilution 1:1000), anti‐DNMT3A (CST, 3598S; dilution 1:1000), anti‐DNMT3L (Abcam, ab3493; dilution 1:2500), anti‐DNMT1 (Abcam, ab19905; dilution 1:1000), anti‐β‐CATENIN (CST, 8480; dilution 1:1000), anti‐H3K27ac (Abcam, ab4729; dilution 1:1000), anti‐H3 (Abcam, ab176842; dilution 1:1000) and anti‐β‐ACTIN (Abcam, ab8227; dilution 1:5000). Horseradish peroxidase‐conjugated secondary antibodies (1:5000) were incubated for 1 hours at room temperature, and proteins were detected by ECL plus reagent. After rinsing with TBST, Clarity™ Western ECL Substrate (BIO‐RAD) was used for visualization, and ChemiDoc™ MP Imaging System (BIO‐RAD) was used for band detection.
6
Histone Extraction and Western Blot Analysis
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Cellular total protein was extracted with radioimmunoprecipitation assay buffer containing protease inhibitors. For histone extraction, cells were collected by refrigerated centrifugation at 300 × g for 10 min. The pellet was incubated for 30 min in hypotonic lysis buffer [10 mM Tris-Cl (pH 8.0), 1 mM KCl, 1.5 mM MgCl2, 1 mM DTT and protease inhibitors]. The nuclei were resuspended in acid-extraction buffer (0.4 N H2SO4), and the extracted histones were precipitated with trichloroacetic acid and resuspended in deionized water. Equal amounts of protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were incubated with primary antibodies, including anti-TBX1 (ab109313, Abcam), anti-H3K4me1 (ab176877, Abcam), anti-H3K4me2 (ab32356, Abcam), anti-H3K4me3 (ab8580, Abcam), anti-histone H3 (ab176842, Abcam) and anti-β-actin (BS6007MH, Bioworld Technology), overnight at 4°C and then with horseradish peroxidase-conjugated secondary antibodies (Sinopept) for 1 h at room temperature. Visualization was performed using an enhanced chemiluminescence detection system (Millipore). Experiments were repeated in triplicate.
7
FGF10 Signaling Pathway Regulation
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FGF10 was obtained from the School of Pharmacy, Wenzhou Medical University (Wenzhou, China). Antibodies against p-Akt (#13038), Bax (#14796), Bcl-2 (#2764), and cleaved-caspase-3 (#9664) were obtained from Cell Signaling Technology. Antibodies against MBP (ab40390), NF200 (ab4680), Nrf2 (ab62352), NQO1 (ab34173), MPZ (ab31851), and Akt (ab179463), Histone H3 (ab176842) and the PI3K/Akt inhibitor LY294002 (ab120243), were purchased from Abcam. Antibodies against GAPDH (10494-1-AP), HO-1 (10701-1-AP), SOD2 (24217-1-AP), and PCNA (10205-2-AP) were purchased from Proteintech. An antibody against S100 (sc-53438) was obtained from Santa Cruz Biotechnology.
8
ChIP-qPCR Analysis of Protein-DNA Interactions
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ChIP experiments were performed with three independent biological replicates using the approach as described previously (Mishra et al., 2007 (link), 2011 (link)). Antibodies used to capture DNA-protein complexes were anti-HA agarose (A2095; Sigma Aldrich), anti-Myc agarose (A7470; Sigma Aldrich), anti-Flag agarose (A2220; Sigma Aldrich), and anti-histone H3 (ab176842; Abcam). ChIP-qPCR was done using Fast SYBR Green Master Mix in 7500 Fast Real Time PCR System (Applied Biosystems, Foster City, CA) following conditions described previously (Mishra et al., 2015 (link)). The enrichment as presented as percent input was determined using the ΔΔCT method (Livak and Schmittgen, 2001 (link)).
9
Histone ChIP-qPCR for H3K4me1
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Histone ChIP was performed as previously described with modifications (11 (link)). Protein A magnetic beads (10002D; Invitrogen), anti-H3 (ab176842; Abcam), and anti-H3K4me1 (ab176877; Abcam) were used. After immunoprecipitation, the eluted DNA was quantified by qPCR with primers listed in SI Appendix, Table S1 . Data were normalized to 1% of input and presented as the ratio of H3K4me1 to H3.
10
Chromatin Immunoprecipitation Assay for p53 and Histone Marks
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ChIP assays to evaluate p53 and histone mark enrichment were performed using the SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technology, Inc., Danvers, MA, USA) following manufacturer’s protocol. The following ChIP-grade antibodies were used: anti-histone H3-acetyl K27, H3 tri-methyl K4, H3 methyl K4, H3 tri-methyl K9 (cat. No. ab4729, ab8580, ab176842, ab176916; Abcam, Cambridge, UK) and anti-p53 antibody DO-1 (sc-126 Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). DNA levels for the regions of interest were quantified by qPCR using specific primers (Supplementary Table S1 ). Details regarding the number of technical and biological replicates are listed in the figure legends.
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