rabbit polyclonal hmgb1 primary antibody  (Abcam)

Journal: Frontiers in Immunology

Article Title: Airway epithelial cell-specific deletion of HMGB1 exaggerates inflammatory responses in mice with muco-obstructive airway disease

doi: 10.3389/fimmu.2022.944772

Figure Lengend Snippet: HMGB1 expression is up-regulated in the BALF of Scnn1b-Tg+ (Tg+) lungs. The representative immunoblots (left) and corresponding quantification of the immunoblots (right). Equal volumes of cell-free BALF from WT and Tg+ adult mice (n=4/group) were used. α-tubulin was used as a loading control. Error bars represent Mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. ** p < 0.01.

Article Snippet: Then, the membrane was incubated with rabbit polyclonal HMGB1 primary antibody (ab18256; Abcam, Cambridge, MA).

Techniques: Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Airway epithelial cell-specific deletion of HMGB1 exaggerates inflammatory responses in mice with muco-obstructive airway disease

doi: 10.3389/fimmu.2022.944772

Figure Lengend Snippet: Airway epithelial cell-specific deletion of HMGB1. (A) Schematic diagram of transgenes used in the generation of airway epithelial cell-specific HMGB1-deficient mice. Airway epithelial cell-specific HMGB1-deficient Scnn1b -Tg+ (CCSP-Cre + / Hmgb1 fl/fl /Tg+) mice were generated by crossing club cell-specific Cre recombinase (CCSP-Cre + ), floxed Hmgb1 ( Hmgb1 fl/fl ), and Scnn1b -Tg+ mice. (B) Immunohistochemistry for HMGB1 in lung sections from airway epithelial cell-specific HMGB1-sufficient and airway epithelial cell-specific HMGB1-deficient WT and Tg+ mice. Red solid arrow indicates the HMGB1-stained cells. Red dotted arrow indicates the cells negatively stained for HMGB1. Bar graph shows percent HMGB1-stained cells in the airways. Sample size n=6/group; Error bars represent Mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. **** p < 0.0001. (C) Western blot showing the depletion of HMGB1 in Cre + / Hmgb1 fl/fl /WT (Cre + /WT) juveniles (red open bar), while comparable HMGB1 between Cre - Hmgb1 fl/fl /Tg+ (Cre - /Tg+) (blue solid bar) and Cre + / Hmgb1 fl/fl /Tg+ (Cre + /WT) (red solid bar) juveniles. Equal volumes of cell-free BALF from all the groups were used as loading samples. M, band from size ladder. Sample size n=7-15/group. Error bars represent Mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Then, the membrane was incubated with rabbit polyclonal HMGB1 primary antibody (ab18256; Abcam, Cambridge, MA).

Techniques: Generated, Immunohistochemistry, Staining, Western Blot

Journal: Frontiers in Immunology

Article Title: Airway epithelial cell-specific deletion of HMGB1 exaggerates inflammatory responses in mice with muco-obstructive airway disease

doi: 10.3389/fimmu.2022.944772

Figure Lengend Snippet: Airway epithelial cell-specific deletion of HMGB1 modulates immune cell recruitment in airspaces of Tg+ mice. Total cell counts (A) are shown for Cre - /WT [blue open bar], Cre + /WT [red open bar], Cre - /Tg+ [solid blue bar], and Cre + /Tg+ [solid red bar] mice). Differential cell counts (B) and the relative percentages (C) are presented as stacked bar graph (macrophages [red bar], neutrophils [blue bar], eosinophils [green bar], and lymphocytes [black bar]). The significant differences are shown by horizontal lines of cell-specific colors (macrophages [red line], neutrophils [blue line], eosinophils [green line], and lymphocytes [black line]). For enhanced clarity in the comparisons, these stacked graphs are replotted for individual cell types ( Supplemental Figure 1 ). Error bars represent Mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Then, the membrane was incubated with rabbit polyclonal HMGB1 primary antibody (ab18256; Abcam, Cambridge, MA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Airway epithelial cell-specific deletion of HMGB1 exaggerates inflammatory responses in mice with muco-obstructive airway disease

doi: 10.3389/fimmu.2022.944772

Figure Lengend Snippet: Airway epithelial cell-specific HMGB1-deficient mice exhibit elevated BALF protein and dsDNA contents in Tg+ mice. The total protein contents (µg/ml) (A) and dsDNA contents (ng/µl) (B) in cell-free BALF from WT mice (with Cre - or Cre + status) and Tg+ mice (with Cre - or Cre + status). Error bars represent Mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. Sample size n=6-9/group. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Then, the membrane was incubated with rabbit polyclonal HMGB1 primary antibody (ab18256; Abcam, Cambridge, MA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Airway epithelial cell-specific deletion of HMGB1 exaggerates inflammatory responses in mice with muco-obstructive airway disease

doi: 10.3389/fimmu.2022.944772

Figure Lengend Snippet: Airway epithelial cell-specific deletion of HMGB1 alters the levels of inflammatory mediators in the airspaces of Tg+ mice. Cell-free BALF cytokine levels (picograms per milliliter) of G-CSF (A) , KC (B) , MIP-2 (C) , MCP-1 (D) , MIP-1α (E) , MIP-1β (F) , IP-10 (G) , and TNF-α (H) in WT mice (with Cre - or Cre + status) and Tg+ mice (with Cre - or Cre + status) are shown. Red dotted horizontal lines indicate the lower limit of detection (LOD) obtained in the assay. The values that were below the LOD were assigned value 0.01 unit less than the LOD. Sample size n=7-9/group. Error bars represent Mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Then, the membrane was incubated with rabbit polyclonal HMGB1 primary antibody (ab18256; Abcam, Cambridge, MA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Airway epithelial cell-specific deletion of HMGB1 exaggerates inflammatory responses in mice with muco-obstructive airway disease

doi: 10.3389/fimmu.2022.944772

Figure Lengend Snippet: Airway epithelial cell-specific deletion of HMGB1 compromises bacterial clearance of Tg+ mice. CFUs were counted in BALF from mice with different genotypes (Cre - /WT [blue open bar], Cre + /WT [red open bar], Cre - /Tg+ [solid blue bar], and Cre + /Tg+ [solid red bar] mice). The CFU values were log 10 -transformed. Error bars represent Mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. ** p < 0.01, **** p < 0.0001.

Article Snippet: Then, the membrane was incubated with rabbit polyclonal HMGB1 primary antibody (ab18256; Abcam, Cambridge, MA).

Techniques: Transformation Assay

Journal: Frontiers in Immunology

Article Title: Airway epithelial cell-specific deletion of HMGB1 exaggerates inflammatory responses in mice with muco-obstructive airway disease

doi: 10.3389/fimmu.2022.944772

Figure Lengend Snippet: Airway epithelial cell-specific deletion of HMGB1 does not alter the airway epithelial cell composition and mucoobstruction. (A) Immunostaining of FOXJ1, CCSP, MUC5AC, and MUC5B in lung sections were performed to check the epithelial cell composition of the four groups (Cre - /WT [blue dotted border], Cre + /WT [red dotted border], Cre - /Tg+ [blue solid border], and Cre + /Tg+ [red solid border] mice). (B) Semiquantitative assessment for mucoobstruction from AB/PAS-stained left lung sections. Absolute quantification of Muc5ac mRNA (C) and Muc5b mRNA (D) in lung tissues. Different groups are shown as: Cre - /WT (blue open bar), Cre + /WT (red open bar), Cre - /Tg+ (blue solid bar), and Cre + /Tg+ (red solid bar). Error bars represent Mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. * p < 0.05, **** p < 0.0001.

Article Snippet: Then, the membrane was incubated with rabbit polyclonal HMGB1 primary antibody (ab18256; Abcam, Cambridge, MA).

Techniques: Immunostaining, Staining

Journal: Critical Care

Article Title: High-mobility group box 1 and the receptor for advanced glycation end products contribute to lung injury during Staphylococcus aureus pneumonia

doi: 10.1186/cc13162

Figure Lengend Snippet: High mobility group box 1 (HMGB1) concentrations in mouse bronchoalveolar lavage (BAL) fluid during S. aureus pneumonia. Wt mice were intranasally infected with 1 × 10 7 colony forming units (CFU) S. aureus and euthanized after 6, 24, 48 and 72 hours. HMGB1 concentrations in BAL fluid were quantified by densometric analysis of HMGB1 western blots and expressed as a percentage of the mean density of control BAL fluid samples (0 hours). Data represent the means ± standard error of the mean (n = 5 mice per time point). * P <0.05 versus naïve mice (0 hours).

Article Snippet: Following blocking with 5% nonfat dry milk proteins (Protifar; Nutricia, Zoetermeer, The Netherlands) in 0.1% Tween 20 PBS (PBS-T), membranes were washed and incubated overnight in 1 μg/ml primary rabbit anti-HMGB1 polyclonal antibody (Abcam, Cambridge, UK) in 1% nonfat dry milk proteins in PBS-T at 4°C.

Techniques: Infection, Western Blot

Journal: Critical Care

Article Title: High-mobility group box 1 and the receptor for advanced glycation end products contribute to lung injury during Staphylococcus aureus pneumonia

doi: 10.1186/cc13162

Figure Lengend Snippet: Anti-high mobility group box 1 (HMGB1)-treated mice show reduced lung pathology early after induction of S. aureus pneumonia. Representative slides of lung H&E staining of control treated (A) and anti-HMGB1 treated mice (C) , original magnification × 2. The boxed areas are also shown at a higher magnification for controls (B) and anti-HMGB1-treated mice (D) , original magnification × 10. Scale bars indicate 200 μm. Total pathology scores were determined at the indicated time points post infection in control treated (gray) and anti-HMGB1 treated mice (white) according to the scoring system described in the Methods section (E) . Total protein was measured in BAL fluid from control (grey) and anti-HMGB1 treated (white) mice (F) . Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (7 to 8 mice per group at each time point). ** P <0.01 versus control treated mice at the same time point.

Article Snippet: Following blocking with 5% nonfat dry milk proteins (Protifar; Nutricia, Zoetermeer, The Netherlands) in 0.1% Tween 20 PBS (PBS-T), membranes were washed and incubated overnight in 1 μg/ml primary rabbit anti-HMGB1 polyclonal antibody (Abcam, Cambridge, UK) in 1% nonfat dry milk proteins in PBS-T at 4°C.

Techniques: Staining, Infection, Whisker Assay

Journal: Critical Care

Article Title: High-mobility group box 1 and the receptor for advanced glycation end products contribute to lung injury during Staphylococcus aureus pneumonia

doi: 10.1186/cc13162

Figure Lengend Snippet: Influx of neutrophils in bronchiolar lavage fluid of control or anti-HMGB1-treated mice

Article Snippet: Following blocking with 5% nonfat dry milk proteins (Protifar; Nutricia, Zoetermeer, The Netherlands) in 0.1% Tween 20 PBS (PBS-T), membranes were washed and incubated overnight in 1 μg/ml primary rabbit anti-HMGB1 polyclonal antibody (Abcam, Cambridge, UK) in 1% nonfat dry milk proteins in PBS-T at 4°C.

Techniques:

Journal: Critical Care

Article Title: High-mobility group box 1 and the receptor for advanced glycation end products contribute to lung injury during Staphylococcus aureus pneumonia

doi: 10.1186/cc13162

Figure Lengend Snippet: Anti - high mobility group box 1 (HMGB1) treatment reduces IL-1β and keratinocyte - derived chemokine (KC) levels in bronchiolar lavage (BAL) fluid after intranasal infection with S. aureus. Cytokine (TNF-α, IL-6 and IL-1β) (A - C) and chemokine (KC and macrophage inflammatory protein (MIP)-2) (D - E) levels in BAL fluid at different time points after intranasal infection of 1 × 10 7 colony-forming units (CFU) S. aureus in mice treated with control (gray) and anti-HMGB1 antibodies (white). Data are expressed as box-and-whisker diagrams depicting the median, the smallest observation, lower quartile, median, upper quartile and largest observation (7 to 8 mice per group at each time point). * P <0.05, ** P <0.01 versus Wt mice at the same time point.

Article Snippet: Following blocking with 5% nonfat dry milk proteins (Protifar; Nutricia, Zoetermeer, The Netherlands) in 0.1% Tween 20 PBS (PBS-T), membranes were washed and incubated overnight in 1 μg/ml primary rabbit anti-HMGB1 polyclonal antibody (Abcam, Cambridge, UK) in 1% nonfat dry milk proteins in PBS-T at 4°C.

Techniques: Derivative Assay, Infection, Whisker Assay

Journal: Critical Care

Article Title: High-mobility group box 1 and the receptor for advanced glycation end products contribute to lung injury during Staphylococcus aureus pneumonia

doi: 10.1186/cc13162

Figure Lengend Snippet: Bacterial outgrowth in Receptor for advanced glycation end products ( Rage ) −/− mice is reduced early after intranasal infection with S. aureus . Bacterial loads after intranasal infection with 1 × 10 7 colony-forming units (CFU) S. aureus in bronchiolar lavage (BAL) fluid (A) of mice treated with control (gray) and anti-HMGB1 antibodies (white) and in BAL fluid (B) of Wt (dark gray), Toll-like receptor ( tlr ) 4 −/− (light gray) and rage −/− mice (white) at 6, 24, 48 and 72 hours after infection. Data are expressed as box-and-whisker diagrams depicting the median, the smallest observation, lower quartile, median, upper quartile and largest observation (n = 7 to 8 mice per group at each time point). ** P <0.01 versus Wt mice at the same time point.

Article Snippet: Following blocking with 5% nonfat dry milk proteins (Protifar; Nutricia, Zoetermeer, The Netherlands) in 0.1% Tween 20 PBS (PBS-T), membranes were washed and incubated overnight in 1 μg/ml primary rabbit anti-HMGB1 polyclonal antibody (Abcam, Cambridge, UK) in 1% nonfat dry milk proteins in PBS-T at 4°C.

Techniques: Infection, Whisker Assay

Journal: International Journal of Molecular Sciences

Article Title: Dietary Antioxidants Significantly Attenuate Hyperoxia-Induced Acute Inflammatory Lung Injury by Enhancing Macrophage Function via Reducing the Accumulation of Airway HMGB1

doi: 10.3390/ijms21030977

Figure Lengend Snippet: Sulforaphane (SFN) decreases the hyperoxia-induced extracellular accumulation of HMGB1 in RAW 264.7 cells. RAW 264.7 cells were exposed to 21% O 2 (white bar) or 95% O 2 (black bar) for 24 h in the presence of various concentrations of SFN (grey bars) (diluted in DMSO as the vehicle). HMGB1 levels in the cell culture supernatant were determined using Western blot analysis. The blot indicates the bands of HMGB1 in each group. Each value represents the mean ± SEM of at least three independent experiments. * p ≤ 0.05 compared to 0 µM SFN vehicle control group. # p ≤ 0.05 compared to 21% O 2 .

Article Snippet: Membranes were washed 3 times with TBST and incubated overnight at 4 °C with anti-HMGB1 rabbit polyclonal primary antibody (#D3E5, Cell Signaling Technologies, Danvers, MA, USA; 1:1000).

Techniques: Cell Culture, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Dietary Antioxidants Significantly Attenuate Hyperoxia-Induced Acute Inflammatory Lung Injury by Enhancing Macrophage Function via Reducing the Accumulation of Airway HMGB1

doi: 10.3390/ijms21030977

Figure Lengend Snippet: Ascorbic Acid (AA) attenuates hyperoxia-induced HMGB1 accumulation in the airways. Mice were exposed to ≥98% O 2 or 21% O 2 for 72 h and randomized to receive either ascorbic acid (50 mg/kg i.p. ) or saline. Animals were sacrificed and BALF was harvested and HMGB1 levels were determined. HMGB1 levels were analyzed using Western blot analysis. Each value represents the mean ± SEM of at least three independent experiments. * p ≤ 0.05 compared to 98% O 2 control mice. # p ≤ 0.05 compared to 21% O 2 control mice.

Article Snippet: Membranes were washed 3 times with TBST and incubated overnight at 4 °C with anti-HMGB1 rabbit polyclonal primary antibody (#D3E5, Cell Signaling Technologies, Danvers, MA, USA; 1:1000).

Techniques: Saline, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Dietary Antioxidants Significantly Attenuate Hyperoxia-Induced Acute Inflammatory Lung Injury by Enhancing Macrophage Function via Reducing the Accumulation of Airway HMGB1

doi: 10.3390/ijms21030977

Figure Lengend Snippet: The proposed mechanism by which antioxidants ascorbic acid (AA) and sulforaphane (SFN) attenuate hyperoxia-induced lung injury and compromised macrophage function in phagocytosis. Prolonged exposure to hyperoxia increases the production of intracellular ROS and HMGB1 release, inhibiting lung macrophage phagocytosis and efferocytosis, leading to an inflammatory response that produces cell injury. The high levels of airway HMGB1 induce the infiltration of leukocytes into the airways, which further release ROS and HMGB1, contributing to a cycle of dysregulated inflammation, augmenting cell injury and leading to lung damage. Supplementation of AA or SFN significantly reduces ROS and inhibits HMGB1 release, attenuating the cycle of cell injury and ameliorating HALI.

Article Snippet: Membranes were washed 3 times with TBST and incubated overnight at 4 °C with anti-HMGB1 rabbit polyclonal primary antibody (#D3E5, Cell Signaling Technologies, Danvers, MA, USA; 1:1000).

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Spinal cord astrocyte identification and high mobility group box-1 (HMGB1) knockdown. a Spinal cord astrocytes were identified using immunofluorescence. The percentage of cells stained with the astrocytic marker S100β, which were identified as astrocytes, was more than 95% of the total cells (three replicates). b HMGB1 knockdown efficiency in the plasma membrane and cytoplasm of spinal cord astrocytes was evaluated using Western blot for HMGB1 protein levels. Results were obtained after 72 h of specific HMGB1 shRNA treatment. HMGB1 protein levels were decreased to approximately 30% of normal levels with shRNA multiplicity of infection 60 as compared to normal astrocytes. * P < 0.05 vs. normal group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Immunofluorescence, Staining, Marker, Western Blot, shRNA, Infection

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of oxygen-glucose deprivation/reoxygenation (OGD/R) on cellular swelling, high mobility group box-1 (HMGB1), and aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes as well as levels of HMGB1 and interleukin-6 (IL-6) released into the surrounding medium. a Astrocyte volume measurement was performed using a Live Cell Imaging System. Cellular volume was calculated by the average value of four measured diameters of the largest compiled Z-slice image. Cellular volumes of spinal cord astrocytes were significantly increased at 2, 6, 12, 24, and 48 h during reoxygenation after OGD when compared with normal astrocytes. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). b Membrane and cytoplasmic HMGB1 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). c Membrane and cytoplasmic AQP4 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). d HMGB1 levels in the surrounding medium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). e IL-6 levels in the surrounding medium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, Live Cell Imaging

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of inhibiting high mobility group box-1 (HMGB1) on cellular swelling in cultured spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R). a Astrocyte volume analysis was performed using a Live Cell Imaging System, and cellular volume was calculated by the average value of four measured diameters. Inhibiting HMGB1 using either HMGB1 shRNA or ethyl pyruvate (EP) significantly blocked increases in cellular volume of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation when compared with astrocytes of the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). b — d Effects of inhibiting HMGB1 on spinal cord astrocytic morphology and ultrastructure were evaluated using transmission electron microscopy at 6, 12, and 24 h during reoxygenation after OGD. After OGD/R, spinal cord astrocytes showed swelling at 6, 12, and 24 h during reoxygenation. The mitochondrial (M) swelling, endoplasmic reticulum (ER) swelling and fragmentation, and an increase in the number of lysosomes (L) were concurrent with this observation. However, astrocytic swelling, mitochondrial (M) swelling, endoplasmic reticulum (ER) swelling and fragmentation, and the increase in lysosome (L) number after OGD/R were reduced by HMGB1 inhibition using either HMGB1 shRNA or EP (× 50,000, bar equal to 1 μm, three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Cell Culture, Live Cell Imaging, shRNA, Transmission Assay, Electron Microscopy, Inhibition

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of inhibiting high mobility group box-1 (HMGB1) on HMGB1, aquaporin-4 (AQP4), and toll-like receptor-4 (TLR4) expression in cultured spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R) as well as levels of HMGB1 and interleukin-6 (IL-6) release into the surrounding medium. a Inhibiting HMGB1 using either HMGB1 shRNA or ethyl pyruvate (EP) significantly suppressed the increased levels of HMGB1 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). b Inhibiting HMGB1 significantly suppressed the increased levels of AQP4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). c Inhibiting HMGB1 significantly suppressed increased levels of TLR4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). d HMGB1, AQP4, and TLR4 immunofluorescence on spinal cord astrocytes at 24 h into the reoxygenation process after OGD showed significantly increased membrane and cytoplasmic levels of HMGB1, AQP4, and TLR4 in the OGD/R group when compared with those in the normal group. These were markedly suppressed in both the OGD/R + HMGB1 shRNA and OGD/R + EP groups (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). e , f Inhibiting HMGB1 mitigated increases in levels of HMGB1 and IL-6 in the surrounding medium when compared with levels in the OGD/R group at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, shRNA, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of either inhibiting high mobility group box-1 (HMGB1) or toll-like receptor-4 (TLR4) on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytic swelling, TLR4, myeloid differentiation primary response gene 88 (MyD88), aquaporin-4 (AQP4) upregulation, and nuclear factor-kappa B (NF-κB) activation as well as levels of interleukin-6 (IL-6) released into the surrounding medium. a Inhibiting HMGB1 (using either HMGB1 shRNA or ethyl pyruvate (EP)) or TLR4 (using CLI-095 or C34) significantly reduced the increase in cellular volume of spinal cord astrocytes at 24 h during the reoxygenation process after OGD when compared with those in the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). b Inhibiting HMGB1 or TLR4 significantly suppressed the increased levels of TLR4, MyD88, and AQP4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 24 h during the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). c Inhibiting HMGB1 or TLR4 significantly suppressed the increased nuclear levels of NF-κB and the upregulation of cytoplasmic p-IκBα in spinal cord astrocytes after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). d Immunofluorescence results showed that either inhibiting HMGB1 or TLR4 decreased membrane and cytoplasmic TLR4 and AQP4 upregulation and attenuated the increases of nuclear NF-κB when compared with the OGD/R group at 24 h during reoxygenation (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). e Inhibiting HMGB1 or TLR4 reduced increased levels of IL-6 in the surrounding medium when compared with those of the OGD/R group after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Activation Assay, shRNA, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of nuclear factor-kappa B (NF-κB) inhibition on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytic swelling, NF-κB activation, and aquaporin-4 (AQP4) upregulation, as well as levels of interleukin-6 (IL-6) released into the surrounding medium. a NF-κB inhibition (using BAY 11-7082) significantly suppressed the increased nuclear levels of NF-κB and the upregulation of cytoplasmic p-IκBα in spinal cord astrocytes after 24 h of the reoxygenation phase after OGD. * P < 0.05 vs. OGD/R group (three replicates). b NF-κB and AQP4 immunofluorescence in spinal cord astrocytes after 24 h of the reoxygenation process after OGD showed significantly increased nuclear levels of NF-κB and membrane and cytoplasmic levels of AQP4 in the OGD/R group. Levels were markedly attenuated in the OGD/R + HMGB1 shRNA, OGD/R + BAY 11-7082, and OGD/R + EP groups (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). c NF-κB inhibition significantly reduced the increase in cellular volume of spinal cord astrocytes at 24 h during the reoxygenation process after OGD when compared with those of the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). d NF-κB inhibition significantly suppressed increased AQP4 levels in both the plasma membrane and cytoplasm of spinal cord astrocytes after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). e NF-κB inhibition reduced increased levels of IL-6 in the surrounding medium when compared with those of the OGD/R group after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Inhibition, Activation Assay, Immunofluorescence, shRNA

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of recombinant HMGB1 (rHMGB1) on aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes. Incubation of cultured spinal cord astrocytes with rHMGB1 (0, 0.1, 1, 10, and 20 ng/ml) for 24 h did not induce dose-dependent increases in the membrane and cytoplasmic AQP4 expression (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Recombinant, Expressing, Cell Culture, Incubation

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of interleukin-6 (IL-6) on aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes. a Spinal cord astrocytes were exposed to exogenous IL-6 at 0, 0.1, 1, or 10 ng/ml. After 24 h exposure, the membrane and cytoplasmic AQP4 expression in spinal cord astrocytes were markedly increased in the IL-6 0.1 ng/ml group, IL-6 1 ng/ml group, and IL-6 10 ng/ml group. * P < 0.05 vs. 0 ng/ml group (three replicates). b IL-6 levels increased in the surrounding medium of the OGD/R group after 24 h of the reoxygenation process after OGD. In comparison, this increase was significantly reduced in the OGD/R + HMGB1 shRNA group. * P < 0.05 vs. OGD/R group (three replicates). c The effects of astrocyte conditioned medium (ACM) on AQP4 expression in cultured spinal cord astrocytes. Twenty-four hours exposure of spinal cord astrocytes to the ACM obtained from the OGD/R group significantly increased the membrane and cytoplasmic AQP4 expression when compared with astrocytes incubated with the ACM obtained from the OGD/R + HMGB1 shRNA group. * P < 0.05 vs. astrocytes + OGD6h/R24h ACM group (three replicates). d Western blot analysis showed that the neutralizing anti-rat-IL-6 antibody could significantly reverse the upregulation effect of exogenous IL-6 or OGD/R ACM containing increased IL-6 on AQP4 expression in cultured spinal cord astrocytes. # P < 0.05 vs. astrocytes + IL-6 group; * P < 0.05 vs. astrocytes + OGD6h/R24h ACM group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, shRNA, Incubation, Western Blot