FAIM-L overexpression, XIAP down-regulation, and their cross-regulation were tested by immunocytochemistry in infected hippocampal primary cultures. Immunocytochemistry was performed using anti-XIAP (1:500; BD Biosciences), anti-FAIM-L (1:500; in house), and rabbit anti-GFP (1:500; Invitrogen), followed by Alexa 568 and Alexa 488-coupled antibodies (1:500; Invitrogen). In DRG neurons, XIAP, FAIM-L, pan-ERK, NF66 and caspase-3 levels were assessed by Western blot using anti-XIAP (BD Biosciences; 1:20,000), anti-FAIM-L (in house; 1:2,000), anti-pan-ERK (BD Biosciences; 1:20,000), anti-NF66 (Covance; 1:10,000), anti-caspase-3 (Cell Signaling; 1:1,000) and Histone H3 (Thermo Scientific; 1:40,000) antibodies.
Histone h3
Manufactured by Thermo Fisher Scientific
Histone H3 is a core histone protein found in eukaryotic cell nuclei. It plays a fundamental role in the packaging and organization of DNA within the chromatin structure. Histone H3 is essential for the regulation of gene expression and chromatin dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Thermo Fisher Scientific (2 mentions)
Anti pan erk, by BD (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
Pan erk, by BD (1 mentions)
Anti xiap, by BD (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Caspase 3, by BD (1 mentions)
Vinculin, by Thermo Fisher Scientific (1 mentions)
Sirt3, by Thermo Fisher Scientific (1 mentions)
Cardiac troponin t, by Thermo Fisher Scientific (1 mentions)
Cox 4, by Bio-Rad (1 mentions)
Actinβ, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
E cadherin, by Thermo Fisher Scientific (1 mentions)
Vimentin, by Thermo Fisher Scientific (1 mentions)
N cadherin, by Thermo Fisher Scientific (1 mentions)
10 protocols using histone h3
1
FAIM-L and XIAP Cross-Regulation in Neurons
2
Antibody Preparation for Immunoblotting
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Antibodies used for immunoblots were purchased from the indicated companies: MST1 (BD-611052 and H00006789-M02), GAPDH (CS-2118), P-LATS (CS-8654), LATS (SC-398560), Histone H3 (CS-9715), cl-CAS3 (CS-9661), cl-CAS9 (CS-9509), Actin-β (SC-69879), SIRT3 (SC-365175 and CS-5490), Vinculin (sc-25336), H2B (CS-12364), Pser14-H2B(CS-6959), Tri-Methyl-Histone (Lys27) H3 (CS-9733), Cardiac Troponin T (Thermo MA5-12,960), COX IV (CS- 4850) [BD = Bio-Rad; CS = Cell Signaling; H = Invitrogen; SC = Santa Cruz]. Anti-mouse and anti-rabbit HRP-linked antibodies were purchased from Bio-Rad and Cell Signaling Technology. All antibodies were diluted in 5% non-fat dry milk powder in 0.05% Tween 20 Tris-buffered saline (TBST) or 3% BSA in TBST.
3
Quantitative Protein Expression Analysis
4
NF-κB Signaling Pathway Analysis
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Western-blot assays were processed to evaluate the expressions of NF-κB (p65) in cytoplasm and nucleus, p-IκBα, IκBα, and active caspase-3. Liver tissues were homogenized immediately after sacrifice to prepare the total protein, nuclear protein, and cytoplasmic protein using Nuclear-Cytoplasm Extraction Kit (Cell Signaling Technology, USA). Protein concentrations were detected by BCA kits (Thermo Scientific, Inc.), and Histone H3 (nuclear protein) and β-actin (cytoplasmic protein) were used as controls. After electrophoresis on 10% SDS-PAGE gels, the proteins were transferred onto the PVDF membrane. After being blocked with 5% skim milk, the membrane was incubated overnight with the primary antibody (Abcam, UK) at 4°C. Subsequently, the blots were washed with TBST, incubated with the secondary antibody at room temperature for 30 min, again washed with TBST, and developed with ECL Plus Western Blot Detection System (Amersham International plc., Buckinghamshire, UK).
5
Protein Expression and Characterization
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Protein levels were quantified by Bradford assay. The protein sample was diluted, heated for denaturation, then subjected to dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene fluoride membranes (PVDF, Millipore). The membrane was blocked in 0.1% Triton X-100 for nuclear proteins and Tween 20 for cytoplasmic proteins, and 5% non-fat milk powder in phosphate-buffered saline for 1 h at 4°C. Primary antibodies against H3K4me1 (ThermoFisher), H3K4me2 ThermoFisher), H3K9me1 (ThermoFisher), H3K9me2 (ThermoFisher), Histone H3 (ThermoFisher), KDM1A (ThermoFisher), Vimentin (ThermoFisher), E-cadherin (ThermoFisher), N-cadherin (ThermoFisher), GAPDH (Abcam), and β-actin (Abcam) were then added, and membranes were kept incubating at 4°C overnight. Corresponding secondary antibodies were applied followed by electrochemiluminescence processing.
6
Histone H3 Binding to Glycated HMGB1
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Fluorescein isothiocyanate (FITC)-labeled human recombinant histone H3 (2 μg, Rockland Immunochemicals Inc., Limerick, PA, USA) was mixed with glycated HMGB1 (2 μg) or human recombinant HMGB1 in Tris-buffered saline (100 μL) and incubated at 4 °C for 12 h. The mixture solution was immunoprecipitated using an anti-HMGB1 antibody (Proteintech) and protein A/G agarose (Santa Cruz) for 3 h at 4 °C. Precipitates were collected by centrifugation and washed five times with lysis buffer. The fluorescence intensity (A520) of histone H3 in the precipitate solution was measured using a fluorescence microplate reader (Thermo Fisher).
7
Western Blot Analysis of Signaling Proteins
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Total cell lysates were obtained as described before [14 (link)] and analyzed by SDS-PAGE using primary antibodies against ABL1 (Santa Cruz Biotechnology, sc-56887), phospho-ABL1 (Cell Signaling, 2865), AML1 (Cell Signaling, 4334), NUP98 (Santa Cruz Biotechnology, sc-101546), phospho-CHK1 (Cell Signaling, 2348), CHK1 (Cell Signaling 2360), phospho-CHK2 (Abcam, ab59408), CHK2 (Cell Signaling, 2662), phospho-S6K (Cell Signaling, 9204), S6K (Cell Signaling, 9202), phospho-AKT (Cell Signaling, 9271), AKT (Cell Signaling, 2920), γ-H2AX (Cell Signaling, 2577), histone H3 (Invitrogen, AHO1432) and β-actin (Santa Cruz Biotechnology, sc-47778). The following secondary antibodies conjugated to HRP were used: goat anti-mouse IgG (EMD Millipore, 12-349) and goat anti-rabbit IgG (EMD Millipore, 12-348).
8
Wnt Signaling in Macrophage-Neuron Crosstalk
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Macrophages treated with MSU and conditioned medium from DRG were lysed in RIPA buffer (Beyotime, P0013C) supplemented with 1 mM PMSF (Beyotime, ST506). We also aliquoted DRG neurons culture supernatant for sFRP2 enzyme-linked immunosorbent assay (ELISA) analyses (LifeSpan Biosciences, LS-F34960). The following antibodies were used to detect proteins. β-catenin (Cell Signaling Technology, 8480, 1:1000), sFRP2 (Abcam, ab86379,1:500), Wnt9a (Abcam, ab125957, 1:1000), FZD2 (Abcam, ab109094, 1:1000), DVL2 (Abcam, ab124933, 1:1000), Cyclin D1 (Cell Signaling Technology, 2978, 1:1000), C-myc (Cell Signaling Technology, 5605, 1:1000), p-IκBα (Cell Signaling Technology, 2859, 1:1000), IκBα (Cell Signaling Technology, 2859, 1:1000), p65 (Cell Signaling Technology, 8242, 1:1000), Histone H3 (Cell Signaling Technology, 4499, 1:1000), GAPDH (Cell Signaling Technology, 5174, 1:1000). Secondary antibodies included goat anti-rabbit IgG (Invitrogen, 35568, 1:30000), goat anti-mouse IgG (Invitrogen, G-21040, 1:30000).
We performed western blotting as previously described 33 (link), and the relative intensities of bands were normalized to those of GAPDH or Histone H3 bands.
We performed western blotting as previously described 33 (link), and the relative intensities of bands were normalized to those of GAPDH or Histone H3 bands.
9
Western Blot Analysis of Diverse Proteins
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Whole-cell lysates prepared using RIPA buffer containing a proteinase inhibitor were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with primary antibodies against Tubulin (Proteintech, 66031-1-Ig), DDX1 (Proteintech, 11357-1-AP), SETD8 (Abcam, ab111691), HistoneH3 (Invitrogen, PA5-16183), CPLX2 (Proteintech, 18149-1-AP), BMAL1 (Invitrogen, PA1-46118), E4BP4 (Proteintech, 11773-1-AP), NR1D1 (Proteintech, 14506-1-AP), EZH2 (Invitrogen, 36-6300), and β-actin (Sigma Aldrich, A1978), followed by the appropriate HRP-conjugated secondary antibodies (Proteintech, SA00001-2/SA00001-1), and protein expression was detected with enhanced luminescence reagents (GE Healthcare, RPN2106).
10
PAK5 Kinase Activity Assay
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The GST-fused proteins were expressed in BL-21 bacteria and purified with GST Sepharose beads (Amersham) and used for PAK5 kinase assay as described previously (10) (link). GST-fusion proteins were purified in vitro and washed three times with kinase buffer (50mM HEPES, pH 7.5, 10mM MgCl2, 2mM MnCl2 and 0.2mM DTT). And afterward for 60min at 30°C with commercialized PAK5 kinase (Cell Signaling Technology) or immunoprecipitated cell synthesized PAK5 kinase domain for kinase assay in 50μl of kinase buffer added with 10μCi of [γ-32 P] ATP (5,000 Ci/mM) and 2.5μM cold ATP. Reactions were stopped by the addition of a 6×SDS loading buffer. After 10% SDS-PAGE and transferred onto PVDF membranes 32 P-labeled proteins were visualized by radioautography with Molecular Imager RX (BIO-RAD). Histone H3 (Invitrogen) was used as a positive control. Ponceau stain indicated the loading amounts of GST-fusion proteins.
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