Authenticated 22Rv1 and DU145 prostate cancer cell lines free of mycoplasma contamination were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and used within 20 passages as previously described (9 (link)). FKA was isolated and purified by LKT Laboratories, Inc. (St. Paul, MN, USA) from the kava root extracts. MLN4924 was purchased from MedChemExpress LLC. (Monmouth Junction, NJ, USA). DMEM/F12, RPMI1640, penicillin–streptomycin, supplement B27 and N2, recombinant human fibroblast growth factor-basic (rhFGF-b), recombinant human epithelial growth factor (rhEGF), accutase, and fetal bovine serum (FBS) were purchased from Fisher Scientific (Hampton, NH, USA). The primary antibodies against Nanog and Keratin 8 (CK8) were obtained from Cell Signaling Technology (Danvers, MA, USA), anti-Sox2 from Life Technology Corporation (Carlsbad, CA, USA), anti-Oct4 from Abcam (Waltham, MA, USA), and anti-β-tubulin from Santa Cruz Biotechnology Inc. (Dallas, TX, USA), respectively.
Anti oct4
Manufactured by Abcam
Sourced in United Kingdom, United States, Germany
Anti-OCT4 is a primary antibody that recognizes the OCT4 (also known as POU5F1) protein. OCT4 is a transcription factor that plays a critical role in the self-renewal of undifferentiated embryonic stem cells and is considered a key marker of pluripotency.
Automatically generated - may contain errors
Lab products found in correlation
Anti sox2, by Abcam (27 mentions)
Anti nanog, by Abcam (25 mentions)
Anti gapdh, by Abcam (8 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Anti β actin, by Abcam (6 mentions)
Anti cd44, by Abcam (5 mentions)
Anti rhoa, by Cell Signaling Technology (5 mentions)
Anti bmi1, by Abcam (4 mentions)
Anti β actin, by Santa Cruz Biotechnology (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Protein lysis buffer, by Merck Group (4 mentions)
Mitochondrial marker antibody sampler kit, by Cell Signaling Technology (4 mentions)
Ecl reagent, by Bio-Rad (4 mentions)
Anti hla g 4h84, by Novus Biologicals (4 mentions)
Anti human prl 1, by Abcam (4 mentions)
Anti cdk4, by Cell Signaling Technology (4 mentions)
Anti rock1, by Cell Signaling Technology (4 mentions)
Anti cyclin d1, by Cell Signaling Technology (4 mentions)
Anti atp5b, by Santa Cruz Biotechnology (4 mentions)
Anti gapdh, by AbFrontier (4 mentions)
74 protocols using anti oct4
1
Prostate Cancer Cell Line Characterization
2
Islet Inflammatory and Dedifferentiation Analysis
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Islets were harvested, fixed in 4% paraformaldehyde, and immersed in 30% sucrose solution to be embedded in a frozen block. Inflammatory markers and dedifferentiation markers were detected in cell sections by immunofluorescence. Anti-FoxO1 (Santa Cruz, Dallas, Texas, USA), anti-cleaved caspase 3 (Cell Signaling, Danvers, MA, USA), anti-Oct4 (Abcam, Cambridge, UK), anti-NGN3 (Abcam), anti-A11 oligomer (Invitrogen), anti-Pdx1 (Abcam), anti-IL-1β (Abcam), and anti-insulin (Abcam) were used as primary antibodies. M1/M2 macrophage markers were identified using the antibodies of M2 macrophage differentiation markers (anti-arginase-1; Cell Signaling) and M1 macrophage differentiation markers (CD80; Abcam). Sections were incubated with primary antibody diluted in blocking buffer overnight at 4°C. Secondary antibodies (Alexa Fluor® 488-conjugated goat anti-rabbit, Alexa Fluor® 488-conjugated goat anti-mouse, and Alexa Fluor® 594-conjugated goat anti-rabbit; Abcam) were added and incubated for 1 h at room temperature. Counterstaining was performed using DAPI (1 : 10,000). Images were obtained from each section using a fluorescent microscope (Olympus, Shinjuku, Tokyo, Japan).
3
Immunophenotypic Characterization of Stem Cells
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The sorted SP and non-SP cells were fixed onto glass slides in ice-cold 4% formaldehyde (4°C, 10 min), and blocked with blocked with 1% bovine serum albumin (Sigma-Aldrich) for 30 min. Slides were incubated with mouse monoclonal anti-CD133 (1:200; cat. no. ab5558; Abcam), anti-Oct-4 (1:100; cat. no. ab59545; Abcam) and anti-ABCG2 (1:100; cat. no. ab3380; Abcam) for 1 h. Following washing the slides with phosphate-buffered saline, slides were incubated with fluorescein isothiocyante-conjugated chicken anti-mouse IgG (1:200; cat. no. ab112455; Abcam) overnight in dark room. Nuclei were counterstained with 4,6-diamidino-2-phenylindole and viewed using a DMI 4000 B fluorescence microscope (Leica, Wetzlar, Germany). All images were processed using Adobe Photoshop, version CS6 (Adobe System, Inc., San Jose, CA, USA).
4
Western Blot Analysis of Stem Cell Markers
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Harvested cells were lysed by RIPA buffer. After sonication, samples were centrifuged at 12 000 g for 15 min at 4°C. Total protein concentration was determined by applying DC Protein Assay Kit I (Bio‐Rad, USA). Proteins were transferred to Hybond nitrocellulose membranes (USA) after separation on 12% SDS‐PAGE. 5% skim milk was added to seal the membrane in Tris‐buffered saline (pH 7.5) at room temperature. The membrane was incubated with the primary antibody overnight at 4°C, followed by 4‐h incubation with the secondary antibody at room temperature. Protein bands were developed by ECL kit (Millipore, USA). Protein levels were assessed by ImageJ (USA). Primary antibodies including anti‐CD133, anti‐CD44, anti‐Oct‐4, anti‐HK2, anti‐PKM2, anti‐LDHA, anti‐β‐actin, and secondary antibody anti‐IgG were purchased from Abcam (UK).16
5
Immunofluorescence Analysis of EMT Markers
6
Quantifying Pluripotency Markers by Western Blot
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Protein was extracted from cells by using RIPA Buffer (Thermo Scientific, Waltham, MA, USA), and subsequently quantified with the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Twenty micrograms of total proteins per lane were separated on a 4–12% Bis-Tris gel, and transferred onto a polyvinylidene difluoride (PVDF) membrane (Thermo Scientific). The membrane was blocked and incubated with primary antibodies overnight at 4 °C (PAX6 monoclonal antibody: Invitrogen, MA1-109, 1:100; anti-OCT4, Abcam, Cambridge, MA, USA, 1:100) followed by secondary antibodies (goat anti-mouse Alexa Fluor Plus 488: Invitrogen, 1:1000; goat anti-rabbit Qdot 605, Invitrogen, 1:50) for 60 min at room temperature. All blots used β-actin (Abcam, 1:500) as a positive control. Three independent Western blot experiments were performed. Blots were imaged using a Fujifilm Fluoro Image Analyzer FLA-5000. Protein band densities were quantified using Fujifilm Science Lab Image Gauge Ver. 4.0.
7
Histone Modifications and Chromatin Interactions
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Rabbit polyclonal to H1, HMGN1, HMGN2, and H3 were from our laboratory, anti H3K27ac (Abcam#ab4729), anti H3K27me3 (Abcam#ab6002), monoclonal anti H1(Milipore-Sigma #05-457), anti CEBPB (Abcam#ab32358), Anti-Brd3 (Active Motif #61489), Anti-Brd4 (Bethyl Laboratories #A301-985A100), Anti-CEBPB (Abcam #ab32358), Anti-CTCF (EMD Millipore #07-729), Anti-Ets1 (Active Motif #39580), Anti-Ikaros (Active Motif #39355), Anti-Irf8 (Bethyl Laboratories #A304-027A), Anti-Klf4 (Abcam #106629), Anti-Nanog (Active Motif #61419), Anti-Oct4 (Abcam #ab19857), Anti-p300 (Active Motif #61401), Anti-Pax5 (Abcam #183575), Anti-Sox2 (Abcam #97959).
The following recombinant mononucleosomes were purchased from Active Motif: unmodified (#81070); H3K27me3 modified (#81834), H3K27ac modified (#81077).
Wild type and HMGN DKO mouse embryonic fibroblasts, embryonic stem cell lines55 (link) and resting B cells56 (link) were as previously described. Peptides Histone H3 (23–34) peptide, KAARKSAPATGG and Histone H3K27ac (23–34) peptide, KAAR - K(Ac)—SAPATGG were from AnaSpec, Inc.
The following recombinant mononucleosomes were purchased from Active Motif: unmodified (#81070); H3K27me3 modified (#81834), H3K27ac modified (#81077).
Wild type and HMGN DKO mouse embryonic fibroblasts, embryonic stem cell lines55 (link) and resting B cells56 (link) were as previously described. Peptides Histone H3 (23–34) peptide, KAARKSAPATGG and Histone H3K27ac (23–34) peptide, KAAR - K(Ac)—SAPATGG were from AnaSpec, Inc.
8
Protein Expression Analysis by Western Blot and Immunofluorescence
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Western blotting and immunofluorescence analyses were performed as published23 (link) with the following antibodies: anti-OCT4 (Abcam, Cambridge, UK), anti-Tra-1-60 (Merck Millipore, Darmstadt, Germany), anti-NANOG (Stemgent, Lexington, USA), anti-SSEA-4 (Merck Millipore, Darmstadt, Germany), anti-TUJ1 (Covance Inc., Princeton, USA), anti-Tbr1 (Abcam, Cambridge, UK), anti-Brn2 (Santa Cruz), anti-ε-sarcoglycan (esg2-1355, published antibody against the brain-specific isoform of the protein)24 (link), anti-FLAG (Sigma Aldrich, St. Louis, USA), anti-Flotilin-1 (Cell Signaling Technology, Danvers, USA), and anti-β-actin (Sigma Aldrich, St. Louis, USA).
9
Stem Cell Marker Expression Profiling
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Following transfection or activator treatment in T25 cell culture vials, nuclear proteins were isolated from TPC-1 and KTC-1 cells treated with LiCl using the NE-PER nuclear extraction kit. The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with PMSF, maintaining a ratio of RIPA to PMSF at 100:1. For the separation of cell lysates, 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed, followed by the transfer of proteins onto nitrocellulose membranes. The blots were incubated using primary antibodies, namely Anti-Oct4 (1:1000), Anti-SOX2 (1:1000), anti-β-catenin (1:1000), Anti-CD133 (1:1000), Anti-ALDH1A1 (1:1000), Anti-EpCAM (1:1000) from Abcam, UK, and Nanog (1:1000), anti-LEF-1 (1:1000) from CST, USA. We used β-actin (1:1000, CST, USA) as a loading control and SKL2001 as a β-catenin activator. Every experiment was replicated three times. For the WB analysis, ImageJ software was utilized to measure the gray value of the bands. The gray value of the target protein was subtracted from the gray value of the internal reference protein, and subsequent normalization was performed.
10
Western Blot Analysis of Stem Cell Markers
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Total proteins were extracted using RIPA lysis buffer on ice, electrophoresed on 12% SDS-PAGE gels (Bio-Rad) and blotted onto nitrocellulose membranes (Amersham Biosciences Corp, Sunnyvale, CA). The membranes were blocked with 5% non-fat milk powder at room temperature for 2 hours and incubated overnight with primary antibodies: anti-ATG4A (1:400) (Abcam, Cambridge, UK), anti-E-cadherin (1:2000) (BD Transduction Laboratories, Franklin Lakes, NJ), anti-N-cadherin (1:1000) (BD Transduction Laboratories), anti-vimentin (1:1000) (BD Transduction Laboratories), anti-Sox2 (1:500) (Abcam), anti-Oct4 (1:400) (Abcam), anti-Bmi-1 (1:400) (Abcam), anti-LC3I/II (1:1000) (Cell Signaling Technology, Danvers, MA) and anti-β-actin antibody (1: 2000) (Sigma-Aldrich). After three 5-min washes, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1: 2000) (Santa Cruz Biotechnology, Dallas, TX) for 2 hours at room temperature and then washed again in TBS-T and visualized with an enhanced chemiluminescence kit (ECL-kit) (Santa Cruz Biotechnology). All of the experiments were performed in triplicate.
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