Total cell lysates (20μg) were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antibodies for CD147 (BD), HO-1, p-H2A.X/t-H2A.X, Cyclophilin-A, HMGB1 (Cell Signaling) and β-Actin (Sigma) for loading controls. Immunoreactive bands were identified using an enhanced chemiluminescence reaction (Perkin-Elmer), and visualized by autoradiography.
Hmgb1
Manufactured by Cell Signaling Technology
Sourced in United States, China
HMGB1 is a highly conserved nuclear protein that plays a key role in the regulation of chromatin structure and transcription. It functions as a DNA-binding protein and can act as a transcriptional regulator, modulating the accessibility of transcription factors to their target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (17 mentions)
Gapdh, by Cell Signaling Technology (10 mentions)
Cleaved caspase 3, by Cell Signaling Technology (7 mentions)
Caspase 3, by Cell Signaling Technology (6 mentions)
Erk1 2, by Cell Signaling Technology (4 mentions)
P jnk, by Cell Signaling Technology (4 mentions)
Gapdh, by Santa Cruz Biotechnology (4 mentions)
Bcl 2, by Cell Signaling Technology (4 mentions)
β actin, by Merck Group (3 mentions)
Las 4000, by Cytiva (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (3 mentions)
P erk1 2, by Cell Signaling Technology (3 mentions)
Phospho nf κb p65, by Cell Signaling Technology (3 mentions)
Myd88, by Cell Signaling Technology (3 mentions)
Nf κb p65, by Cell Signaling Technology (3 mentions)
β catenin, by Cell Signaling Technology (3 mentions)
Cleaved caspase 1, by Cell Signaling Technology (3 mentions)
Nf κb, by Cell Signaling Technology (3 mentions)
Mmp 2, by Cell Signaling Technology (3 mentions)
81 protocols using hmgb1
1
SDS-PAGE Immunoblot Analysis
2
Colocalization of HMGB1 and Exosomes in AECs
3
Immunoblotting Analysis of Phospho-Akt and HMGB1
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PVAT was homogenized in a lysis buffer (2% SDS, 4 M Urea, 1 mM EDTA, 150 mM NaCl, 50 mM Tris pH 8.0). Immunoblotting was performed with a phospho (Ser473)-Akt (9271, Cell Signaling Technology, Danvers, MA, USA), a total Akt antibody (9272, Cell Signaling Technology), HMGB1 (6893, Cell Signaling Technology), β-actin (4970, Cell Signaling Technology). Immunoblots were detected and analyzed with ECL Prime Western Blotting Detection Reagent and ImageQuant LAS 4000 mini (GE Healthcare, Little Chalfont, UK).
4
Colchicine Mitigates CCl4-Induced Liver Fibrosis
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In this study, colchicine was purchased from Xishuangbanna Banna Pharmaceutical Co. Ltd. (Yunnan, China). CCl4 was supplied by Tianjin Guangfu Chemical Research Institute (Tianjin, China). Alanine amino transferase (ALT, Cat. No.: 20180627), aspartate amino transferase (AST, Cat. No.: 20180607), collagen IV (COL-IV, Cat. No.: 201809), hyaluronic acid (HA, Cat. No.: 201808), laminin (LN, Cat. No.: 201809), and precollagen type III (PC III, Cat. No.: 201809) detection kits were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The antibodies of phospho-c-Jun, phospho-MEK1/2, HMGB1, α-SMA, phospho-ERK1/2, and ERK1/2 were bought from Cell Signaling Technology (United States). The antibodies of MEK1/2 and COL-I were purchased from Abcam (United States) and the antibodies of RAGE and GAPDH were bought from ABclonal (China).
5
Amnion-Derived Cell Culture and Transfection
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The primary hAECs from amnion tissues were plated and maintained in culture at 37 °C in a humidified incubator in an atmosphere of 5% CO2. JetPrime transfection reagent was purchased from PolyPlus-Transfection (Strasbourg, France). Antibodies to HMGB1 (Cell Signaling Technology, Beverly, MA, USA) and Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. LPS from Escherichia coli strain 0111:B4 was obtained from Sigma-Aldrich.
6
Western Blot Analysis of Apoptosis Markers
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Cells were lysed using RIPA buffer supplemented with aprotinin (Sigma), and Halt Protease Inhibitor Cocktail (100X) (Thermo Scientific). Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Bio-Rad, Mississauga, ON). Primary antibodies used in this study include caspase-1 (Sant Cruz Biotechnology, Dallas, TX), cleaved caspase-1 (AdipoGen Life Science, San Diego, CA), caspase-3 (Sant Cruz Biotechnology, Dallas, TX), cleaved caspase-3 (Cell signaling technology, Danvers, MA), HMGB1 (Cell signaling technology, Danvers, MA), MLKL (Abcam, Boston, MA), phosphorylated MLKL (Abcam, Boston, MA), CRT (Abcam, Boston, MA), and GAPDH (Sant Cruz Biotechnology, Dallas, TX). The expression levels Relative expression of proteins were visualized using the corresponding secondary HRP-conjugated antibodies and Amersham ECL select western blotting detection reagent, as described previously (46 (link)).
7
Protein Expression Analysis in GH4C1 Cells
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Western blotting was used to assess MYC, BAX, BCL-2, HMGB1, SOX7, and IL-16 levels in GH4C1 cells 48 hr after transfection with miRNA oligos. Briefly, equal amounts of protein (40 μg) were separated by 12% SDS-PAGE, transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA), and then blocked in 5% milk in Tris-buffered saline (TBS)-Tween 20 (0.5%). Proteins were then probed with rabbit antibodies against human MYC, BCL2, SOX7, HMGB1, BAX, AND IL-16 (Cell Signaling Technology, Danvers, MA, USA) and corresponding peroxidase-conjugated secondary antibodies (Cell Signaling Technology) and visualized using enhanced chemiluminescence (ECL) solution (Santa Cruz Biotechnology, Dallas, TX, USA). For Cell Signaling Technology c-MYC (#13987), BAX (#2772), HMGB1 (#6893), and GAPDH (#2118), and for Abcam BCL-2 (ab196495), SOX7 (ab89954), and IL-16 (ab180792), the dilution ratio was 1:1,000.
8
Western Blot Analysis of HMGA Proteins
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Cell lysates were obtained using a lysis buffer containing phosphatase inhibitor and the protease inhibitor phenylmethanesulfonyl fluoride (100 mM; cat. no. KGP250; Nanjing KeyGen Biotech Co., Ltd.) and the protein concentration in the lysates determined by Bradford assay. A total of 100 µg lysate was separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane at 250 mA for 1 h. The PVDF membranes were blocked with 5% FBS for 1.5 h at 37°C and incubated overnight at 4°C with rabbit anti-mouse antibodies against high mobility group (HMG)A1 (1:10,000; cat. no. ab129153; Abcam), HMGA2 (1:1,000; cat. no. D1A7; Cell Signaling Technology, Inc.), HMGB1 (1:10,000; cat. no. ab79823; Abcam), HMGB2 (1:10,000; cat. no. ab124670; Abcam) and β-actin (1:1,000; cat. no. BM0627; Wuhan Boster Biological Technology, Ltd.). The PVDF membranes were washed three times with TBST (0.1% Tween) and incubated with goat anti-rabbit Immunoglobulin G-HRP (1:1,000; cat. no. BA1054; Wuhan Boster Biological Technology, Ltd.) for 1.5 h at 37°C, followed by the 3,3′-diaminobenzidine (EMD Millipore) method at room temperature for 15 sec. PVDF membranes were subjected to densitometry analysis (chemiDox.XRS+; Bio-Rad Laboratories, Inc.), then the image was analyzed using Quantity One 4.0 software (Bio-Rad Laboratories, Inc.).
9
Immunoblotting Analysis of PD-L1, STAT3, and HMGB1
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Cells were lysed in RIPA buffer containing proteinase inhibitors. Equal amount of proteins was separated by a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. Following electrophoresis, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, blocked in 5% non-fat milk, and incubated with primary antibodies at 4°C overnight. Antibodies against PD-L1, STAT3, p-STAT3, and HMGB1 were purchased from Cell Signaling Technology (Louis Park, MN, USA). After washing with Tris-buffered saline + Tween 20 (TBS/T) three times, membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Bioworld Technology) at room temperature for 2 h. The protein bands were visualized by enhanced chemiluminescence. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the loading control.
10
Protein Extraction and Western Blot Analysis
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Whole cell protein extraction, cytosolic protein extraction, and nuclear protein extraction were extracted according to previous studies (Pan et al., 2014 (link); Zhang et al., 2016a (link)). Equal protein amounts were separated by polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked in 5% skim milk for 2 h at room temperature and then incubated with primary antibodies against caspase-3 (1:400, cat #9661, Cell Signaling), SIRT1 (1:200, cat #SC-15404, Santa Cruz), HMGB1 (1:1000, cat #3935S, Cell Signaling), TLR4 (1:200, cat #sc-30002, Santa Cruz), Myd88 (1:200, cat #sc-11356, Santa Cruz), Nf-κB p65 (1:200, cat #sc-372, Santa Cruz), β-actin (1:3000, cat #AP0060, Bioworld Technology, Minneapolis, MN, USA), and Histone H3 (1:3000, cat #BS7416, Bioworld Technology) overnight at 4°C. The membrane was incubated with horseradish peroxidase (HRP)-conjugated IgG for 2 h at room temperature. Detection was performed by enhanced chemiluminescence solution (Thermo Fisher Scientific, Waltham, MA, USA). Band density was quantified with UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA).
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