Cytoplasmic and nuclear extracts were prepared according to standard protocols [36 (link)] and the Bradford assay method (Bio-Rad) was used to determine protein concentration. Then, 20 μg of both cytoplasmic and nuclear extracts were separated by SDS-PAGE. Antibodies against PAD4 (1:1000; Abcam), citrullinated histone H3 (CitH3) (1:1000; Abcam) and nuclear factor (NF)-κB (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were dispensed overnight (4 °C) and peroxidase-conjugated secondary antibody was administered for 1 h (RT). The immunoreaction was visualized using ECL plus (GE Healthcare, Buckinghamshire, UK). Stain-free technology (Bio-Rad) was used as loading protein control and densitometric analysis was performed with Image Lab software (Bio-Rad).
Citrullinated histone h3 cith3
Manufactured by Abcam
Sourced in United States
Citrullinated histone H3 (CitH3) is a post-translational modification of histone H3 protein, where arginine residues are converted to citrulline. This modification is involved in various biological processes, such as gene regulation and chromatin remodeling. CitH3 can be used as a marker for the detection and study of these cellular mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (1 mentions)
Bradford assay method, by Bio-Rad (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Nf κb, by Santa Cruz Biotechnology (1 mentions)
Stain free technology, by Bio-Rad (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
P gsk3β ser9, by Cell Signaling Technology (1 mentions)
Gapdh, by Signalway Antibody (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Confocal microscopy, by Zeiss (1 mentions)
Lyg6 antibodies, by Thermo Fisher Scientific (1 mentions)
3 protocols using citrullinated histone h3 cith3
1
Quantification of Citrullinated Histone H3
2
Western Blotting Antibody Panel
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Western blotting was carried out as previously described [17 (link)], with the following antibodies: Mcl-1 (1:1000; Genetex, USA), cleaved-caspase 3 (1:1000; Cell Signaling Technology, USA), p-GSK-3β (Ser9) (1:1000; Cell Signaling Technology, USA), citrullinated histone H3 (Cit-H3, 1:1000; Abcam, USA), and GAPDH (1:5000; Signalway Antibody, Maryland, USA). ImageJ was used to calculate the grayscale values.
3
Detecting Citrullinated Histone H3 in Neutrophil Extracellular Traps
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Synovial tissues were digested in Hank's Balanced Salt Solution (Thermo Fisher Scienti c, Waltham, MA, USA) containing 0.25% trypsin (Gibco) at 37°C. Treated neutrophils or embedded tissues were xed in 4% paraformaldehyde for 15 min and permeabilized in 0.01% Triton X-100 for 10 min at room temperature.
Samples were then stained with citrullinated histone H3 (CitH3) (1:50 dilution; Abcam, Cambridge, UK) and LYG6 antibodies (1:100 dilution; eBioscience, San Diego, CA, USA). The nucleus was counter-stained with DAPI. Images were captured by confocal microscopy (Zeiss, Jena, Germany) from ve randomly elds. The results were expressed as the number of NETs/50 Ly6G-positive cells.
Samples were then stained with citrullinated histone H3 (CitH3) (1:50 dilution; Abcam, Cambridge, UK) and LYG6 antibodies (1:100 dilution; eBioscience, San Diego, CA, USA). The nucleus was counter-stained with DAPI. Images were captured by confocal microscopy (Zeiss, Jena, Germany) from ve randomly elds. The results were expressed as the number of NETs/50 Ly6G-positive cells.
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