Journal: Frontiers in Neuroscience

Article Title: Annexin A1 Bioactive Peptide Promotes Resolution of Neuroinflammation in a Rat Model of Exsanguinating Cardiac Arrest Treated by Emergency Preservation and Resuscitation

doi: 10.3389/fnins.2019.00608

Figure Lengend Snippet: Modulation of indices of neuroinflammation after EPR by experimental group. (A,B) High mobility group protein B1 (HMGB1) expression in brain homogenates from ANXA1sp-treated animals were significantly reduced compared to vehicle (controls). (C,D) Cerebral levels of tumor necrosis alpha (TNFα) and interleukin 6 (IL-6) were increased in the vehicle controls, and reduced with systemic administration of ANXA1sp. (E) Cerebral interleukin 10 (IL-10) was significantly reduced after induction of profound hypothermia (EPR + vehicle) and restored with administration of ANXA1sp. Data presented as mean ± SD ( n = 3–7/group), ∗ P < 0.05 and ∗∗ P < 0.01 compared to vehicle controls or naïve animals, analyzed by one-way ANOVA with Tukey’s multiple comparisons test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Western blotting was performed using SDS–PAGE 4–20 and 8–16% gels gradient gels (Bio-Rad) with the following antibodies: rabbit anti-HMGB1 (Bioss Antibodies Inc., Woburn, MA, United States), rabbit polyclonal antibodies against SIRT3 (28-kDa isoform, Cell Signaling and Abcam), FOXO3a (Cell Signaling), Mn-SOD (Santa Cruz), cleaved caspase-3 (Cell Signaling), Bax and Bcl-2 (Santa Cruz), rabbit anti-LC3B (cell signaling), mouse anti-p62/SQSTM1 (R&D Systems, Minneapolis, MN, United States), and rabbit monoclonal antibody against GAPDH (Cell Signaling Technology, Boston, MA, United States).

Techniques: Expressing

Journal: Brain, behavior, and immunity

Article Title: Alcohol Exposure after Mild Focal Traumatic Brain Injury Impairs Neurological Recovery and Exacerbates Localized Neuroinflammation

doi: 10.1016/j.bbi.2014.11.006

Figure Lengend Snippet: Diagram of coronal rat brain section showing the site of injury and photographed region (4 A). Representative immunofluorescence images (10 ×) of ipsilateral cortex GFAP, ED-1, and HMGB1 immunoreactivity (green) combined with DAPI (blue) at 14 days post-TBI (10 days post-alcohol exposure) (4 B).

Article Snippet: Anti-HMGB1 Rabbit polyclonal (Abcam, Cambridge, UK) , 1:100 , Donkey anti-Rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA) , 1:200.

Techniques: Immunofluorescence

Journal: Brain, behavior, and immunity

Article Title: Alcohol Exposure after Mild Focal Traumatic Brain Injury Impairs Neurological Recovery and Exacerbates Localized Neuroinflammation

doi: 10.1016/j.bbi.2014.11.006

Figure Lengend Snippet: Quantification of ipsilateral cortex GFAP, ED-1, and HMGB1 immunoreactivity at 14 days post-TBI (10 days post-alcohol exposure). Images are quantified as % area of positive staining in 1.035 mm2 (10×). Values are shown as means ± SEM. For GFAP and ED-1, Sham/Air n = 4, Sham/Alcohol n = 4, TBI/Air n = 4, TBI/Alcohol n = 6. For HMGB1, Sham/Air n = 4, Sham/Alcohol n = 5, TBI/Air n = 5, TBI/Alcohol n = 7 (each n represents the average of 3 images taken from each animal) * p < 0.05 of the TBI groups vs. time-matched sham controls; $ p < 0.05 of the TBI/Alcohol group vs. TBI/Air group, by two-way ANOVA (5 A, 5 C, and 5 E).

Article Snippet: Anti-HMGB1 Rabbit polyclonal (Abcam, Cambridge, UK) , 1:100 , Donkey anti-Rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA) , 1:200.

Techniques: Staining

Journal: Brain, behavior, and immunity

Article Title: Alcohol Exposure after Mild Focal Traumatic Brain Injury Impairs Neurological Recovery and Exacerbates Localized Neuroinflammation

doi: 10.1016/j.bbi.2014.11.006

Figure Lengend Snippet: Primary and Secondary Antibodies.

Article Snippet: Anti-HMGB1 Rabbit polyclonal (Abcam, Cambridge, UK) , 1:100 , Donkey anti-Rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA) , 1:200.

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: Differential location of HMGB1 expression in HDFs and KFs. ( a ) HMGB1 expression levels by immunofluorescence. HMGB1 expression (red) was mainly detected in the HDF nuclei. ( b ) HMGB1 expression was observed in KFs, but was more abundant in the cytosol and extracellular space. ( c and d ) HMGB1 protein expression. The levels of proteins were assessed by western blot. α-tubulin was used as a loading control for cytosol; histone was as a nuclei protein control. Relative levels of HMGB1 in the cytoplasmic fraction were significantly elevated in the KFs. ( e and f ) Stimulation of HDFs with LPS (100 ng/mL) for 24 h and 48 h resulted in increased time-dependent expression of HMGB1 protein in the cell lysate (** p < 0.01). ( g ) Whole cell lysates were fractionated and the levels of HMGB1 in the cytoplasmic and nuclear fractions were determined by western blotting to determine whether LPS treatment induced HMGB1 translocation. HMGB1 translocated from nucleus to cytoplasm in HDFs under LPS treatment (500 ng). Dermal fibroblasts cell line was purchased as primary cell line from the ATCC (American Type Culture Collection, Manassas, VA, USA). Primary keloid fibroblast cell line were obtained from human keloid explant under IRB protocol. Both cells were used for experiment after 2~3 passage.

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: Expressing, Immunofluorescence, Western Blot, Translocation Assay

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: Role of HMGB1 as a profibrotic molecule. ( a ) An MTT assay revealed a significant increase in time-dependent proliferation activity in the HMGB1-treated HDFs (50 and 100 ng; ** p < 0.01). ( b ) Type I collagen mRNA expression was equivalently increased in the HDFs treated with HMGB1 (100 ng) or TGF-β1 (10 ng) (* p < 0.05, ** p < 0.01). There was also an additive increase of type I collagen mRNA expression after simultaneous treatment with HMGB1 and TGF-β1 ( § p < 0.05). ( c ) Levels of collagen types I and III were detected by western blotting in HDF lysates treated with HMGB1 (100 ng) for 24 and 48 h. ( D ) The protein expression levels of collagen types I and III increased significantly after 24 h (** p < 0.01). ( e) The protein expression level of α-SMA was assessed by western blotting in HDF cells treated with HMGB1 (50 and 100 ng). ( f ) Stimulation of HDFs with HMGB1 resulted in increased protein expression of α-SMA (** p < 0.01).

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: MTT Assay, Activity Assay, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: HMGB1 induced TGF-β1, Smads, Erk 1/2, Akt, and NF-κb in HDFs. ( a – c ) Effects of HMGB1 on TGF-β1 signaling pathway in vitro . Significant increases in the expression levels of TGF-β1, Smad 2, and Smad 3 mRNA were observed in the HMGB1 (50 ng)-treated HDFs (** p < 0.01). ( d ) Effects of HMGB1 on TGF-β and phosphor-Smad 2/3 complex protein expressions. ( e , f ) TGF-β and phosphor-Smad 2/3 complex protein levels were significantly increased in the HMGB1 (100 ng)-treated HDFs (** p < 0.01). ( g ) Western blotting analysis in normal dermal spheroids. Increased expression of TGF-β and phosphor-Smad 2/3 complex protein were observed in normal dermal spheroids treated with HMGB1 (400 ng and 1000 ng). ( h ) The levels of Erk 1/2, Akt, and NF-κb protein were detected by western blotting in HDFs lysates treated with HMGB1 (50 ng and 100 ng). ( i to k ) Erk 1/2, Akt, and NF-κb protein levels in HMGB1 (100 ng)-treated HDFs were significantly increased by 1.5-, 2.0-, and 2.3-fold, respectively, versus non-treated HDFs (** p < 0.01).

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: In Vitro, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: HMGB1 reduced MMP 1 and TIMP-1 mRNA expression. HDFs were treated with 50 ng/mL of HMGB1 for various times, and examined using real-time RT-PCR. ( a and c ) MMP-1 mRNA levels were significantly reduced, but TIMP1 mRNA level was increased in 48 hr after treatment with HMGB1 (** p < 0.01). ( b ) MMP-2 mRNA expression increased up to 24 h, but the increase was not statistically significant.

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: Effect of HMGB1 on normal dermal spheroids. ( a ) Picrosirius red staining of HMGB1-treated normal dermal spheroids. Collagen deposition increased. The HMGB1 dose dependency of dense and coarse collagen bundles was investigated. ( b ) Semi-quantitative analysis revealed that treatment with 400 and 1000 ng of HMGB1 significantly increased collagen deposition in normal dermal spheroids by 2.9- and 2-fold, respectively, versus non-treated dermal spheroids (** p < 0.01). ( c ) Western blotting analysis of HMGB1-treated normal dermal spheroids for the detection of type-I and -III collagen protein expression level ( d ) Immunohistochemical staining of HMGB1-treated normal spheroid sections for the detection of types I and III collagen, elastin, and fibronectin protein. Increased expression of ECM components (collagen types I and III, elastin, and fibronectin protein) was examined versus nontreated spheroids. Original magnification: 400×. ( e to h ) Semi-quantitative image analysis of ECM protein expression. The expression levels of type I collagen, type III collagen, elastin, and fibronectin increased significantly versus nontreated spheroids (* p < 0.05; ** p < 0.01).

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: Staining, Western Blot, Expressing, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: Immunohistochemical analysis of HMGB1 and its receptors (RAGE and TLR4) in human keloid tissues. ( a ) Higher expression levels of HMGB1 and its receptors (RAGE and TLR4) were observed in the region of the keloid tissues than in the adjacent normal tissue. Original magnification: 400×. ( b to d ) The comparison of HMGB1, RAGE, and TLR4 expression levels between keloid tissues and extra-lesional normal tissue (** p < 0.01).

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: Immunohistochemical staining, Expressing

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: A mild heat shock (HS) pretreatment attenuates oxidative stress (H2O2)-induced HMGB1 release in macrophages cultures. A, Effects of mild or severe heat shock on HMGB1 release. RAW 264.7 cells were subjected to a brief heat shock (1 h) at 42.5°C (mild heat shock), 43.5°C or higher (severe heat shock), after which was returned to 37°C and incubated for additional 12 h. HMGB1 in the culture medium were detected by Western blotting analysis (top). In parallel, the cell viability was determined by LDH release assay (bottom). Ctrl, control cells. Blot is representative of three experiments with similar results. Values are mean ± SEM (n = 3) of three experiments in duplicate. B, Mild HS pretreatment induced Hsp expression. RAW 264.7 cells were subjected to heat shock (42.5°C, 1 h), and cellular levels of Hsp90 and Hsp72 were detected by Western blotting analysis at the indicated time points after heat shock. In parallel, levels of HMGB1 in the culture medium were determined by Western blotting analysis. Tubulin was used as a loading control. Values are representative of three independent experiments with similar results. C, Effects of oxidative stress on Hsp72 expression. RAW 264.7 cells were stimulated with H2O2 at nontoxic (0.125 mM), or low-toxic (0.25 mM) concentrations, and cellular Hsp72 were detected at 12 h poststimulation by Western blotting analysis. GAPDH was used as a loading control. Values are representative of three independent experiments with similar results. D, Mild heat shock pretreatment inhibited H2O2-induced HMGB1 release. After mild heat shock (42.5°C, 1 h), cells were allowed to recover for 12 h at 37°C and then stimulated for 12 h with H2O2 at nontoxic (0.125 mM) or low-toxic (0.25 mM) concentrations. Levels of HMGB1 in the culture medium were determined by Western blotting and expressed (in arbitrary units; AU) as mean ± SEM of three experiments in duplicate. In parallel, the cell viability was determined by MTT assay, and expressed as mean ± SEM (n = 4) of three experiments in duplicate. *, p < 0.05.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Incubation, Western Blot, Lactate Dehydrogenase Assay, Control, Expressing, MTT Assay

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Overexpression of Hsp72 renders macrophages resistant to H2O2-induced HMGB1 release. A, Western blotting analysis of Hsp72 levels in RAW 264.7 cells transfected with empty plasmid (lane 1), Hsp72 expression construct (lane 2) or challenged with mild heat shock (HS, 42.5°C, 1 h). Tubulin was used as a loading control. B, Visualization of fluorescent Hsp72 protein in RAW 264.7 cells transfected with Hsp72 expression construct (top), or empty plasmid (bottom). Nuclei were visualized by Hoechst staining. C, Western blotting analysis of H2O2-induced HMGB1 release in RAW 264.7 cells transfected with empty plasmid, or Hsp72 expression construct. RAW 264.7 cells were stimulated with H2O2 (0.125 and 0.25 mM) for 12 h, and HMGB1 levels in the culture medium were determined, and expressed as mean ± SEM of three experiments in duplicate. *, p < 0.05.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Expressing, Construct, Control, Staining

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Overexpression of Hsp72 attenuates H2O2-induced HMGB1 cytoplasmic translocation in macrophage cultures. A and B, H2O2 induces transient nuclear translocation of Hsp72 in macrophages cultures. pcDNA3.1-Hsp72-transfected RAW 264.7 cells were stimulated with H2O2 (0.125 mM) for various period of time and examined for Hsp72 subcellular localization by immunocytochemistry (A) or cell fractionation/Western blot (B). Green, Hsp72; blue, nuclei. Original magnification, ×400. A nuclear protein, proliferating cell nuclear Ag, was used as a loading control. C, Heat shock induces Hsp72 expression and nuclear translocation in macrophages cultures. RAW 264.7 cells subject to mild heat shock (42.5°C, 1 h), and nuclear Hsp72 content was determined by Western blotting. D, Overexpression of Hsp72 attenuates H2O2-induced HMGB1 cytoplasmic translocation. RAW 264.7 cells transfected with empty pcDNA3.1 plasmid, or pcDNA3.1-Hsp72 construct were stimulated with H2O2 (0.125 mM) for 12 h, and the subcellular localization of HMGB1 was determined by immunocytochemical analysis (D, left). Relative fluorescence intensity of HMGB1 in the nuclear (N) and cytoplasmic (C) regions of multiple representative cells was assayed using the ImageProPlus software (D, right). Image is representative of three experiments with similar results. Red, HMGB1; blue, nuclei. Original magnification, ×1000. Values are means ± SEM (n = 50) of three experiments in duplicate. *, p < 0.05.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Over Expression, Translocation Assay, Transfection, Immunocytochemistry, Cell Fractionation, Western Blot, Control, Expressing, Plasmid Preparation, Construct, Fluorescence, Software

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Hsp72 and HMGB1 are coimmunoprecipitated after oxidative stress or heat shock. A, Coimmunoprecipitation of Hsp72 and HMGB1 from lysate of RAW 264.7 cells. RAW 264.7 cells transfected with pcDNA3.1-Hsp72 construct were stimulated with H2O2 (0.125 mM) for 3 h, and whole-cell lysate was immunoprecipitated with various Abs: nonspecific rabbit serum (Sr), nonspecific mouse serum (Sm), or Abs specific for Hsp72 or HMGB1, respectively. In parallel experiment, RAW 264.7 cells were subjected to mild heat shock (42.5°C for 1 h) and returned to 37°C for 6 h, and whole-cell lysate was immunoprecipitated with HMGB1- or Hsp72-specific Abs, respectively. The precipitated complexes were separately immunoblotted with Hsp72- or HMGB1-specific Abs. IP, immunoprecipitation; IB, Immunoblotting. Hsp72-transfected cell lysate (L) was used as a positive control. Blot is representative of three experiments with similar results. B, Coimmunoprecipitation of Hsp72 and HMGB1 from lysate of human leukemia K562 cells. K562 cells were subjected to oxidative stress (by stimulating withH2O2, 0.125 mM, for 12 h) or mild heat shock (HS, 42.5°C, 1 h, and then returned to 37°C for 6 h), and whole-cell lysate was immunoprecipitated with HMGB1-specific Abs. The precipitated complexes were then sequentially blotted with Hsp72- or HMGB1-specific Abs, respectively. IP, immunoprecipitation; IB, immunoblotting; Ctrl, control cells. Blot is representative of two experiments with similar results. C, Coimmunoprecipitation of Hsp72 and HMGB1 from nuclear and cytoplasmic fraction of RAW 264.7 cells transfected with pcDNA3.1-Hsp72. Cells were subjected to oxidative stress (H2O2, 0.125 mM for 3 h; H), or mild heat shock (42.5°C for 1 h, then returned to 37°C for 6 h; HS), and nuclear or cytoplasmic proteins were immunoprecipitated with Hsp72-or HMGB1-specific Abs. The precipitated complexes were then blotted with Hsp72- or HMGB1-specific Abs. Hsp72-transfected cell lysates was used as a positive control. C, control cells. Blot is representative of two experiments with similar results. D, Stoichiometry analysis of Hsp72-bound HMGB1 in nuclear fractions. RAW 264.7 cells transfected with pcDNA3.1-Hsp72 were stimulated with H2O2 (0.125 mM) for 3 h, and levels of HMGB1 in the whole-cell extracts before (1, set at 100%) or after (2) immunoprecipitation with excessive amount of Hsp72-specific Ab was determined by Western blotting. Blot is representative of three experiments with similar results.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Positive Control, Control

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: An herb-derived antioxidant, quercetin, attenuates H2O2-induced Hsp72 nuclear translocation, and Hsp72-HMGB1 interaction in macrophage cultures. RAW 264.7 cells transfected with pcDNA3.1-Hsp72 construct were pretreated with quercetin (50 μM) for 4 h and subsequently stimulated with H2O2 (0.125 mM) for 3 h. Subcellular localization of Hsp72 in cells was determined by immunocytochemistry (A) or Western blot (B). Results are representative of three experiments with similar results. Green, Hsp72; blue, nuclei. Original magnification, ×400). Ctrl, control cells; Q, + quercetin; H, + H2O2; QH, + quercetin + H2O2. In parallel experiments, whole-cell lysate was immunoprecipitated with Hsp72-specific Abs, and the precipitated complexes were immunoblotted with Hsp72- or HMGB1-specific Abs, respectively (C). IP, immunoprecipitation; IB, immunoblotting. Blot is representative of three experiments with similar results.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Derivative Assay, Translocation Assay, Transfection, Construct, Immunocytochemistry, Western Blot, Control, Immunoprecipitation

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Identification of functional domains of Hsp72 for interacting with nuclear HMGB1. A, Schematic diagram of Hsp72 and two mutants lacking the NLS or PBD. B, Detection of HMGB1 and Myc-tagged Hsp72, Hsp72-ΔNLS, or Hsp72-ΔPBD by Western blotting. RAW 264.7 cells were transiently transfected with pcDNA3.1-Hsp72, pcDNA3.1-Hsp72-ΔNLS, or pcDNA3.1-Hsp72-ΔPBD and stimulated with H2O2 (0.125 mM) for 3 h; nuclear extract was assayed for HMGB1 and Myc-tagged proteins by Western blotting (Immunoblotting; IB) analysis. Blots are representative of two independent experiments with similar results. C, Coimmunoprecipitation of HMGB1 with Myc-tagged Hsp72, Hsp72-ΔNLS, or Hsp72-ΔPBD. In parallel experiments, nuclear extracts were immunoprecipitated (IP) with HMGB1-specific Abs, and the precipitated complexes were then assayed for levels of HMGB1 or Myc-tagged proteins by Western blotting. Blots are representative of two independent experiments with similar results. D, Western blotting of H2O2-induced HMGB1 release (D, left) and translocation (D, right) in RAW 264.7 cells transfected with empty plasmid, pcDNA3.1-Hsp72, pcDNA3.1-Hsp72-ΔNLS, or pcDNA3.1-Hsp72-ΔPBD. Tubulin was used as a loading control. Blots are representative of three independent experiments with similar results.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Functional Assay, Western Blot, Transfection, Immunoprecipitation, Translocation Assay, Plasmid Preparation, Control

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Hypothetical role of Hsp72 in the regulation of oxidative stress-induced HMGB1 cytoplasmic translocation and release. In response to stresses (e.g., heat shock), Hsp72 is produced to maintain a pool of Hsp72 in the cytoplasm. Upon stimulation with secondary oxidative stress (e.g., H2O2), Hsp72 is translocated into the nucleus, where it directly, or indirectly, interacts with various nuclear proteins (such as HMGB1 and histone deacetylase 1 (HDAC1). The intranuclear Hsp72-HMGB1 consequently prevents HMGB1 cytoplasmic translocation and subsequent release via the secretory lysosome pathway.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Translocation Assay, Produced, Histone Deacetylase Assay

Journal: Mediators of Inflammation

Article Title: Elevation of High-Mobility Group Protein Box-1 in Serum Correlates with Severity of Acute Intracerebral Hemorrhage

doi: 10.1155/2010/142458

Figure Lengend Snippet: Serum level of HMGB1 increased significantly in patients with acute ICH. Data are presented as mean ± standard deviation. (a) ELISA assay of the changes of HMGB1 serum levels in patients. (b) A representative picture of Western blot assay showing the changes of HMGB1 serum levels in patients. GAPDH was chosen as the internal control. The changes of HMGB1 were expressed as the ratio of the optical density values of HMGB1 band to the optical density values of GAPDH band. **versus control; P < .01; ## versus good outcome group; P < .01.

Article Snippet: The ECL membranes were incubated with the primary antibodies including mouse antihuman HMGB1 (for HMGB1 assay in serum, 1 : 1000, Santa Cruz) or rabbit antimouse HMGB1 (for HMGB1 assay in brain tissue, 1 : 5000, Santa Cruz), followed by incubation with peroxidase-conjugated secondary antibodies (1 : 2000, Jingmei, China).

Techniques: Standard Deviation, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Mediators of Inflammation

Article Title: Elevation of High-Mobility Group Protein Box-1 in Serum Correlates with Severity of Acute Intracerebral Hemorrhage

doi: 10.1155/2010/142458

Figure Lengend Snippet: The level of HMGB1 increased significantly in the perihematomal tissue of ICH mice. Data are expressed as mean ± standard deviation. (a) ELISA assay of the changes of HMGB1 levels in the perihematomal tissue of ICH mice. (b) A representative picture of Western blot assay showing the changes of HMGB1 levels in the perihematomal tissue of mice. GAPDH was chosen as the internal control. The changes of HMGB1 were expressed as the ratio of the optical density values of HMGB1 band to the optical density values of GAPDH band. n = 15. **versus control; P < .01; ## versus 72 hours; P < .01.

Article Snippet: The ECL membranes were incubated with the primary antibodies including mouse antihuman HMGB1 (for HMGB1 assay in serum, 1 : 1000, Santa Cruz) or rabbit antimouse HMGB1 (for HMGB1 assay in brain tissue, 1 : 5000, Santa Cruz), followed by incubation with peroxidase-conjugated secondary antibodies (1 : 2000, Jingmei, China).

Techniques: Standard Deviation, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Mediators of Inflammation

Article Title: Elevation of High-Mobility Group Protein Box-1 in Serum Correlates with Severity of Acute Intracerebral Hemorrhage

doi: 10.1155/2010/142458

Figure Lengend Snippet: Heme could stimulate the secretion of HMGB1 by cultured microglia. Data are expressed as mean ± standard deviation, n = 6. (a) Microglia were cultured with various concentrations of heme (0 μ M, 10 μ M, 20 μ M, and 30 μ M) for 24 hours. Thereafter, the culture medium was replaced, and the cells were further cultured for 12 hours before collection of the supernatant for ELISA determination of HMGB1 levels. **versus 0 μ M; P < .01; ## versus 10 μ M, 20 μ M; P < .01. (b) Microglia were treated with 30 μ M of heme for various lengths of time (0 h, 4 h, 8 h, 24 h, and 48 h). Thereafter, the culture medium was replaced, and the cells were further cultured for another 12 hours before collection of the supernatant for ELISA determination of HMGB1 levels. **versus 0 h; P < .01; ## versus 4 h, 8 h, or 24 h; P < .01. (c) Microglia were treated with 30 μ M of heme, 30 μ M of FeCl 3 , or 30 μ M of FeSO 4 for 24 hours. Thereafter, the culture medium was replaced, and the cells were further cultured for another 12 hours before collection of the supernatant for ELISA determination of HMGB1 levels. **versus control; P < .01.

Article Snippet: The ECL membranes were incubated with the primary antibodies including mouse antihuman HMGB1 (for HMGB1 assay in serum, 1 : 1000, Santa Cruz) or rabbit antimouse HMGB1 (for HMGB1 assay in brain tissue, 1 : 5000, Santa Cruz), followed by incubation with peroxidase-conjugated secondary antibodies (1 : 2000, Jingmei, China).

Techniques: Cell Culture, Standard Deviation, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancers

Article Title: Loss of PGRMC1 Delays the Progression of Hepatocellular Carcinoma via Suppression of Pro-Inflammatory Immune Responses

doi: 10.3390/cancers13102438

Figure Lengend Snippet: Loss of Pgrmc1 aggravates liver injury while suppressing compensatory proliferation. ( A ) WT and Pgrmc1 -null mice were injected with high-dose DEN (200 mg/kg, i.p.), and their plasma and livers were collected after 48 h. ( B ) Plasma level of alanine aminotransferase (ALT). ( C ) Expression of high mobility group box 1 (HMGB1) in the plasma. The expression level was normalized to that of plasma albumin and expressed relative to the WT group. ( D ) TUNEL staining in the livers of WT and Pgrmc1 -null mice. TUNEL-positive cells were counted and expressed relative to the WT group. Scale bar, 100 μm. ( E ) Ki67 immunostaining in the livers of WT and Pgrmc1 -null mice. Ki67-positive cells were counted and expressed relative to the WT group. Scale bar, 100 μm. ( F ) mRNA expression levels of C-Myc , Cyclin D , and Hgf in the livers of WT and Pgrmc1 -null mice. The expression level was normalized to that of Rplp0 and expressed relative to the WT group. * p < 0.05 in Student’s t -test. All full blot images were provided in .

Article Snippet: The following primary antibodies were used: β-actin (sc-130656, Santa Cruz Biotechnology), phosphor-IκBα (pIκBα), NF-κB p65, phosphor-NF-κB p65 (pNF-κB p65) (9936, CST, MA, USA), EGFR (A2909, ABclonal, MA, USA), phosphor-EGFR (9789, CST, MA, USA), HMGB1 (CSB-PA01604A0Rb, Cusabio, TX, USA), PGRMC1 (rabbit monoclonal, 13856, CST, MA, USA), and IκBα (mouse monoclonal, 9936, CST, MA, USA).

Techniques: Injection, Clinical Proteomics, Expressing, TUNEL Assay, Staining, Immunostaining