Human HMGB1/HMG-1 Antibody

Detection of Human HMGB1/HMG‑1 by Western Blot. Western blot shows lysates of HepG2 human hepatocellular carcinoma cell line, Jurkat human acute T cell leukemia cell line, and HEK293 human embryonic kidney cell line. PVDF membrane was probed with 0.05 µg/mL of Mouse Anti-Human HMGB1/HMG-1 Monoclonal Antibody (Catalog # MAB1690) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for HMGB1/HMG-1 at approximately 26 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of HMGB1/HMG‑1-regulated Genes by Chromatin Immunoprecipitation. HepG2 human hepatocellular carcinoma cell line was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. HMGB1/HMG-1/DNA complexes were immunoprecipitated using 5 µg Mouse Anti-Human HMGB1/HMG-1 PE-conjugated Monoclonal Antibody (Catalog # MAB1690) or control antibody (Catalog # MAB004) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Mouse IgG Secondary Antibody (Catalog # BAF007). Immunocomplexes were captured using 50 µL of MagCellect Streptavidin Ferrofluid (Catalog # MAG999) and DNA was purified using chelating resin solution. TheAFPpromoter was detected by standard PCR.

Detection of HMGB1 in HCT‑116 Human Cell Line by Flow Cytometry. HCT-116 human colorectal carcinoma cell line was stained with Mouse Anti-Human HMGB1/HMG-1 Monoclonal Antibody (Catalog # MAB1690, filled histogram) or isotype control antibody (Catalog # MAB0041, open histogram), followed by Phycoerythrin-conjugated Anti-Mouse IgG F(ab')₂Secondary Antibody (Catalog # F0102B). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005). View our protocol for Staining Intracellular Molecules.

HMGB1/HMG‑1 in CCD‑18Co Human Cell Line. HMGB1/HMG-1 was detected in immersion fixed CCD-18Co human colonic fibroblast cell line using Mouse Anti-Human HMGB1/HMG-1 Monoclonal Antibody (Catalog # MAB1690) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red, upper panel; Catalog # NL007) and counterstained with DAPI (blue, lower panel). Specific staining was localized to nuclei. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

HMGB1/HMG-1 in Human Breast Cancer Tissue. HMGB1/HMG-1 was detected in immersion fixed paraffin-embedded sections of human breast cancer tissue using Mouse Anti-Human HMGB1/HMG-1 Monoclonal Antibody (Catalog # MAB1690) at 5 µg/mL overnight at 4 °C. Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (brown; Catalog # VC001) and counterstained with hematoxylin (blue). Specific staining was localized to cell nuclei. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Detection of Rat HMGB1/HMG-1 by Western Blot The effect of corticosterone on high-mobility group box-1 (HMGB1) release in primary cultured cortical astrocytes. (A) Increasing concentrations of corticosterone induced HMGB1 release from astrocytes. Cells were treated with the indicated concentrations of corticosterone for 24 h, and the amount of HMGB1 in the media was analyzed by Western blotting. Representative blots are shown (HMGB1: 30 kDa). The data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01 vs. basal (concentration “0”) (Dunnett’s test; n = 7–15). (B) Astrocytes were treated with 1 μM corticosterone for the indicated periods of time and the amount of HMGB1 in the media was analyzed by Western blotting. Representative blots are shown (HMGB1: 30 kDa). The data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01 vs. basal (time “0”) (Dunnett’s test; n = 3–15). (C) Astrocytes were treated with 1 μM corticosterone for 24 h, and media concentrations of HMGB1 were analyzed by ELISA. The data are expressed as the mean ± SEM. * p < 0.05 vs. vehicle (t-test; n = 5). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/32344830), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Rat HMGB1/HMG-1 by Western Blot The effect of corticosterone on high-mobility group box-1 (HMGB1) release in primary cultured cortical astrocytes. (A) Increasing concentrations of corticosterone induced HMGB1 release from astrocytes. Cells were treated with the indicated concentrations of corticosterone for 24 h, and the amount of HMGB1 in the media was analyzed by Western blotting. Representative blots are shown (HMGB1: 30 kDa). The data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01 vs. basal (concentration “0”) (Dunnett’s test; n = 7–15). (B) Astrocytes were treated with 1 μM corticosterone for the indicated periods of time and the amount of HMGB1 in the media was analyzed by Western blotting. Representative blots are shown (HMGB1: 30 kDa). The data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01 vs. basal (time “0”) (Dunnett’s test; n = 3–15). (C) Astrocytes were treated with 1 μM corticosterone for 24 h, and media concentrations of HMGB1 were analyzed by ELISA. The data are expressed as the mean ± SEM. * p < 0.05 vs. vehicle (t-test; n = 5). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/32344830), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human HMGB1/HMG-1 by Immunohistochemistry RAGE and HMGB1 are expressed in myofibroblasts of endarterectomised chronic thromboembolic tissues of CTEPH patients.One representative patient is shown (12 out of 15 patients (80%) displayed analogous staining patterns. Photograph showing the macroscopic aspect of a representative PEA specimen (A). Scale bar: 6 cm. Immunohistochemical expression of RAGE (B, scale bar: 20 µm), HMGB1 (C), vimentin (D), alpha-smooth muscle actin (E) and CD34 (F) on adjacent tissue sections of the PEA specimen shown in (A). Scale bar: 40 µm. RAGE receptor for advanced glycation endproducts, HMGB1 high mobility group box 1, CTEPH chronic thromboembolic pulmonary hypertension, PEA pulmonary endarterectomy. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0106440), licensed under a CC-BY license. Not internally tested by R&D Systems.