All general chemicals and reagents were procured from Sigma-Aldrich/ Merck Millipore, HiMedia and Promega. Tissue culture plastic ware was purchased from Jet Biofil or Tarsons India Pvt. Ltd. and Corning Inc. siRNAs were obtained from Dharmacon as siGENOME SMART-pool reagents against Yy1, Prmt5, Notch1, Itch. Oleic acid, HRP-tagged anti-β-ACTIN (A3854), 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) were procured from Sigma-Aldrich. Anti-cleaved NOTCH1, anti-ITCH, anti-YY1, anti- α-TUBULIN, anti-PRMT5, anti-EZH2, anti-H3K27me3 antibodies were procured from Cell Signaling Technology (USA). Anti-H4R3me2s antibody was sourced from Abcam. Anti-ADRP and anti-CD36 antibodies were procured from Santa Cruz Biotechnology (USA). Anti-LAMINB1 antibody was purchased from IMGENEX. HRP conjugated anti-rabbit IgG/anti-mouse IgG was obtained from Jackson ImmunoResearch (USA). Lipofectamine 3000 was purchased from Thermo Fisher Scientific. BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene lipid stain was from Molecular Probes (Invitrogen/Thermo Fisher Scientific).
Anti yy1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-YY1 is a primary antibody that detects the YY1 transcription factor. YY1 is a ubiquitous zinc finger protein that functions as a transcriptional regulator, capable of activating or repressing target genes.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Sigenome smartpool reagent, by Horizon Discovery (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Anti h3k27me3, by Cell Signaling Technology (1 mentions)
Anti cleaved notch1, by Cell Signaling Technology (1 mentions)
Anti itch, by Cell Signaling Technology (1 mentions)
Anti prmt5, by Cell Signaling Technology (1 mentions)
Anti ezh2, by Cell Signaling Technology (1 mentions)
Anti v5 tag antibody, by Thermo Fisher Scientific (1 mentions)
Anti wdr5, by Fortis Life Sciences (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Chemiluminescence system, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Peroxidase hrp conjugated secondary antibody, by Proteintech (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions)
8 protocols using anti yy1
1
Comprehensive Lipid Metabolism Regulatory Assay
2
In Situ Proximity Ligation Assay of MuSCs
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The in situ proximity ligation assay (PLA) was performed on fixed primary proliferating MuSCs using the DuoLink PLA fluorescence technology (Sigma‐Aldrich#DUO92101), following the manufacturer′s protocol. About 2,000 isolated muscle satellite cells were seeded per well of a 96‐well plate and grown to a confluence of about 80%. Cells were fixed using 4% PFA for 7 min at room temperature. Myoblasts were then permeabilized with 0.3% Triton X‐100 in PBS three times for 5 min at room temperature. After two washing steps with PBS, cells were incubated with blocking solution in a humid chamber for 60 min at 37°C followed by incubation with primary antibodies for 1 h at room temperature. The assay was always performed with pairs of antibodies produced in mouse or rabbit, diluted 1:5,000 in antibody diluent. An anti‐V5 tag antibody (Thermo Scientific; 37–7,500) recognizing the endogenous V5‐tagged INO80 was used in combination with anti‐YY1 (Cell Signaling#46395), anti‐WDR5 (Bethyl#A302‐429A), and anti‐Ruvbl2 (Bethyl#A302‐536A) antibodies, respectively. PLA probe incubation, ligation, and signal amplification were performed according to the manufacturer's protocol. After two washing steps with PBS, DAPI was diluted 1:5,000 in PBS and added to the cells for 5 min at room temperature.
3
Protein Expression Analysis by Western Blot
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Total cellular proteins were isolated by incubating the cells with RIPA buffer containing 1% PMSF (Sigma) on ice for 30 minutes. The protein concentrations were quantified by BCA Protein Assay Kit (Beyotime). Proteins were electrophoresed by 10% SDS‐PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were then blocked with 5% skimmed milk for 2 hours at room temperature. Subsequently, membranes were incubated with the primary antibodies, including anti‐YY1 (63227, Cell Signaling Technology 1:1000) and anti‐β‐actin (3700, Cell Signaling Technology 1:1000) for overnight at 4°C. Next morning, the membrane was incubated with peroxidase (HRP)‐conjugated secondary antibody (Proteintech, 1:5000) for 1 hour at room temperature. Finally, the intensity of the bands was detected by the chemiluminescence system (Bio‐Rad) and analysed by Image Lab software.
4
ChIP Assay Protocol for Studying YY1
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A commercially available kit (Beyotime, Jiangsu, China) was used for the ChIP assay. Briefly, cells were treated with 1% formaldehyde for crosslinking and then sonicated on ice. Anti-YY1 (#46395, Cell Signaling Technology) or anti-IgG (#2729, Cell Signaling Technology) antibodies were added to the stained chromatin and incubated overnight. The precipitated chromatin DNA was recovered and analyzed by qRT-PCR. The primer sequences are shown in Table S2 .
5
Western Blot Analysis of Apoptosis Markers
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Proteins were extracted from the cells using radioimmunoprecipitation assay buffer (Invitrogen), and then, the sample concentration was assessed using a bicinchoninic acid protein quantification kit (Thermo Fisher Scientific). Equal amounts of proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA), transferred to polyvinylidene fluoride membranes (Millipore, Temecula, CA), blocked with 5% skim milk, and then incubated with the following primary antibodies: anti-YY1 (#46395, Cell Signaling Technology, Danvers, MA, USA, 1:1000), anti-PARP (#9542, Cell Signaling Technology, 1:1000), anti-Caspase-3 (#9662, Cell Signaling Technology, 1:1000), and anti-GAPDH (#5174, Cell Signaling Technology, 1:1000). Next, the membranes were incubated with secondary antibodies. Protein signals were observed using enhanced chemiluminescence (Bio-Rad), with GAPDH as an internal control (Olympus, Tokyo, Japan).
6
Protein Expression Analysis by Western Blot
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Protein extracts were prepared using RIPA buffer (Sigma: 150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 50 mM Tris, pH8.0) completed with Protease Inhibitor (PI) Cocktail tablet (Sigma). Protein concentration was determined with the Pierce BCA protein assay kit (Thermo Scientific). Protein extracts (20-40 µg) were fractionated by SDS polyacrylamide gel electrophoresis on 4–12% Bis–Tris precast polyacrylamide gels (Invitrogen, Paisley, UK) and transferred to a nitrocellulose membrane (Invitrogen). Bands were visualized by chemiluminescence (ECL Plus; GE Healthcare, Hatfield, UK). Protein quantification was performed using ImageJ and statistical significance was assessed using GraphPad Prism 8. Anti-IRF2 (Abcam, cat#ab124744, 1:500 dilution), anti-IRF9 (Cell Signaling, cat#76684, 1:500 dilution), anti-YY1 (Cell Signaling, cat#2185, 1:500 dilution), anti-SNAI2 (Abcam, cat#ab27568, 1:200 dilution), and anti-p16 (CDKN2A, Abcam, cat#ab54210, 1:1000 dilution) antibodies were used.
7
Western Blot Analysis of Protein Expression
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Proteins were resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a nitrocellulose membrane (0.2 μm pore size) (Bio-Rad). The blots were probed with the following primary antibodies: anti-HSF1 (#4356, polyclonal), anti-Fas (#4233, monoclonal: C18C12), anti-Lamin A/C (#4777, monoclonal: 4C11; for nuclear extracts), anti-Caspase 3 (#9668, monoclonal: 3G2) and anti-H2AUb (Lys 119) (#8240, monoclonal: D27C4) from Cell Signaling Technology (Danvers, MA, USA); anti-YY1 (#sc-7341, monoclonal: H-10), anti-SP1 (#sc-420, monoclonal: 1C6), anti-Ub (rabbit polyclonal, kindly provided by Prof. A. L. Haas, Louisiana State University, Health Sciences Center, New Orleans), anti-β-Actin (#sc-47778, monoclonal: C4; for whole extracts) and anti-β-catenin (#sc-7963, monoclonal: E-5) from Santa Cruz Biotechnology (Dallas, TX, USA); anti-GAPDH (#A300-641A-T, polyclonal; for cytosolic extracts) from Bethyl Laboratories (Montgomery, TX, USA). Immunoreactive bands were detected by horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad). Peroxidase activity was detected with the enhanced chemiluminescence detection method (WesternBright ECL, Advasta, Menlo Park, CA, USA) using the ChemiDoc MP Imaging System (Bio-Rad). Quantification of the protein bands was performed using Image Lab analysis software version 5.2.1 (Bio-Rad).
8
Antibody Characterization and Validation
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The following antibodies were used throughout this study: anti-FKBP3 from Thermo Fisher (catalog no. PA5-19483); anti-HDAC1 (catalog no. ab280198), anti-HDAC2 (catalog no. ab219053), anti-H3K4ac (catalog no. ab176799), and anti-H3K18ac (catalog no. ab40888) from Abcam (Cambridge, UK); and anti-YY1 (catalog no. 46395) and anti-β-actin (catalog no. 4970) from Cell Signaling Technology (MA, USA). We purchased 2× Taq master mix (catalog no. P112) and high-fidelity PCR enzyme 2× Phanta Max master mix (catalog no. P515) from Vazyme (Nanjing, China). PMD18-T (catalog no. 6011) was purchased from TaKaRa (Beijing, China). Cell genome extraction kit (catalog no. DP304) and plasmid extraction kit (catalog nos. DP103, DP108, and DP117) were purchased from Tiangen (Beijing, China). Gel extraction kit (catalog no. CW2302) was purchased from CWBio (Nanjing, China). Luciferase and NanoLuc detection kits (catalog nos. E6110 and N1110, respectively) were purchased from Promega (Madison, USA). Recombinant human IFN-α (catalog no. 11200-1), recombinant human IFN-β (catalog no. 8499-IF), and recombinant human IFN-γ (catalog no. 285-IF) were purchased from R&D Systems (USA). Cell counting kit (CCK-8) and TUNEL apoptosis detection kit (fluorescein isothiocyanate [FITC]) were purchased from Yeasen (Shanghai, China).
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