Journal: Mediators of Inflammation

Article Title: Cooling and Sterile Inflammation in an Oxygen-Glucose-Deprivation/Reperfusion Injury Model in BV-2 Microglia

doi: 10.1155/2021/8906561

Figure Lengend Snippet: Western blot analysis analyzing extracellular proteins and DAMPs. (a) HMGB1, (b) Hsp70, (c) CIRBP, and (d) RBM3 presented as x -fold change relative to Normoxia 37°C control and (e) representative immunoblots at respective timepoints. Cells were exposed to 2 h and 6 h of oxygen-glucose deprivation (OGD, 0.2% O 2 in glucose/serum-free medium) and incubated at 37°C or cooled 1 h after experimental start to 33.5°C. Data from at least 3 individual experiments are presented as mean ± SD. Statistical analysis was conducted using one-way analysis of variance (ANOVA) with Tukey posttest; ∗ p < 0.05 for group comparison and # p < 0.05 for comparison to Normoxia 37°C were considered significant. Panels show representative Immunoblots.

Article Snippet: Primary antibodies for β -Actin (1 : 20000, Cell Signaling, Cat#4967), Caspase 1 (1 : 1000, Cell Signaling, Cat#67314), Caspase 8 (1 : 1000, Cell Signaling, Cat#9429), Caspase 9 (1 : 1000, Cell Signaling, Cat#9508), Caspase 3 (1 : 1000, Cell Signaling, Cat#9662), Hsp70 (1 : 1000, Cell Signaling Technology, Cat#4872) HMGB1 (1 : 2000, Chondrex, Cat#7028), CIRBP (1 : 1000, Abclonal, Cat#A6080), and RBM3 (1 : 1000, Proteintech, Cat#14363-1-AP) were diluted in blocking solution and incubated overnight at 4°C.

Techniques: Western Blot, Incubation

Journal: Gene therapy

Article Title: Measles virus causes immunogenic cell death in human melanoma

doi: 10.1038/gt.2011.205

Figure Lengend Snippet: The inflammatory response associated with MV infection. A; Cytokine/chemokine release. Cell-free supernatants were collected 48 hours after infection with MV and cytokine levels determined by ELISA. Data shown are representative of three independent experiments. B; HMGB1 release. Melanoma cells were treated with MV at MOI from 0.01 to 5. 48hours after infection cell-free supernatant was collected then analysed by western blot for HMGB1. Lanes with protein markers (M) and untreated controls (C) are indicated. Data shown are representative of two separate experiments.

Article Snippet: The supernatants were diluted 1:1 with Laemmli buffer prior to loading 20μL of diluted supernatant on a 10% SDS page gel using standard protocols; staining for HMGB1 was performed using a monoclonal mouse anti-human HMGB1 antibody (R&D systems, Abingdon UK) used at 1μg/mL in a 1:1 mix of Odyssey blocking buffer (LiCor Biotechnology, Cambridge UK) and PBS/0.1% Tween.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Open Life Sciences

Article Title: Aggravated renal fibrosis is positively associated with the activation of HMGB1-TLR2/4 signaling in STZ-induced diabetic mice

doi: 10.1515/biol-2022-0506

Figure Lengend Snippet: Activation of the HMGB1/TLR2/4 signaling pathway in the kidneys of STZ-induced diabetic mice. Mice were treated with STZ at 100 or 150 mg/kg by intraperitoneal injection. The kidney was dissected on Day 64. Immunohistochemical staining of HMGB1, TLR2, and TLR4 in the kidneys (a), photographed at 400× magnification. Protein expression of HMGB1 and p-NF-κB in the kidneys by Western blot (b), n = 3. PCNA and β-actin were used as internal controls. Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, vs the normal group, tested by one-way ANOVA and Fisher’s PLSD.

Article Snippet: After quenching endogenous peroxidase activity with 3% H 2 O 2 and blocking with 5% bovine albumin, the sections were incubated with antibodies recognizing HMGB1 (1:400, Proteintech), TLR2 (1:500, Arigo), TLR4 (1:400, Abcam), F4/80 (1:300, Servicebio), CD14 (1:500, Servicebio), Col IV (1:250, Abcam), and FN (1:250, Abcam) overnight.

Techniques: Activation Assay, Injection, Immunohistochemical staining, Staining, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Anti-HMGB1 mAb Therapy Reduces Epidural Hematoma Injury

doi: 10.3390/ijms25115889

Figure Lengend Snippet: Experimental protocol and changes in HMGB1 levels in the brain and plasma 48 h after the EDH. ( A ) Experimental protocol of the EDH. ( B ) Typical appearance picture of the EDH. The brain samples were collected 48 h after the induction of the EDH from the dotted area in ( B ) and were used for western blotting for HMGB1. ( C ) The representative results of western blotting are shown. A decrease in HMGB1 levels in the ipsilateral cortex (peri-hematoma) in the control IgG group is shown. ( D ) Quantitative analyses of western blotting results were performed using NIH Image J software V.1.53, and the results are expressed as mean ± SE of 3–5 rats per group. Differences were considered significant at # p < 0.05 compared to the sham group. * p < 0.05 compared to the control IgG-treated group. ( E ) Plasma levels of HMGB1 in rats with an EDH were determined by ELISA. Blood samples were collected 48 h after the induction of hemorrhage. The results are expressed as mean ± SE of 3–5 rats per group. # p < 0.05 compared to the sham group. * p < 0.05 compared to the control IgG-treated group.

Article Snippet: The membranes were blocked with 5% non-fat dry milk and then incubated overnight at 4 °C with primary antibodies against HMGB1 (ab18256, Abcam, Waltham, MA, USA), Heat Shock Protein 27 (Hsp27) (ab2790, Abcam, Waltham, MA, USA), Hsp40 (ab69402, Abcam, Waltham, MA, USA), Hsp60 (ab46798, Abcam, Waltham, MA, USA), Hsp90 (ab13492, Abcam, Waltham, MA, USA), Peroxiredoxin 5 (Prx5) (ab180587, Abcam, Waltham, MA, USA), Prx6 (ab195045, Abcam, Waltham, MA, USA), Aquaporin-4 (AQP4) (ab9512, Abcam, Waltham, MA, USA), Protease-Activated Receptor 1 (PAR1) (PA5-116040, Invitrogen, Carlsbad, CA, USA), and Plasminogen Activator Inhibitor-1 (PAI-1) (ab66705, Abcam, Waltham, MA, USA). β-actin (sc-47778, Santa Cruz, CA, USA) was used as a reference protein.

Techniques: Western Blot, Software, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Anti-HMGB1 mAb Therapy Reduces Epidural Hematoma Injury

doi: 10.3390/ijms25115889

Figure Lengend Snippet: Effects of the anti-HMGB1 mAb on the expression of inflammation-related molecules and apoptosis 48 h after the EDH. ( A ) The mRNA expression of TNF-α, iNOS, and IL-1β was measured by quantitative real-time polymerase chain reaction in the cerebral cortex (peri-hematoma) 48 h after the EDH. The primer information refers to previous research . The results are expressed as mean ± SE of 5 rats ( A ). # p < 0.05 compared to the sham group. * p < 0.05 and compared to the control IgG-treated group. ( B ) TUNEL staining was performed to reveal apoptotic cells in the ipsilateral cerebral cortex in each group. Scale bar: 50 μm.

Article Snippet: The membranes were blocked with 5% non-fat dry milk and then incubated overnight at 4 °C with primary antibodies against HMGB1 (ab18256, Abcam, Waltham, MA, USA), Heat Shock Protein 27 (Hsp27) (ab2790, Abcam, Waltham, MA, USA), Hsp40 (ab69402, Abcam, Waltham, MA, USA), Hsp60 (ab46798, Abcam, Waltham, MA, USA), Hsp90 (ab13492, Abcam, Waltham, MA, USA), Peroxiredoxin 5 (Prx5) (ab180587, Abcam, Waltham, MA, USA), Prx6 (ab195045, Abcam, Waltham, MA, USA), Aquaporin-4 (AQP4) (ab9512, Abcam, Waltham, MA, USA), Protease-Activated Receptor 1 (PAR1) (PA5-116040, Invitrogen, Carlsbad, CA, USA), and Plasminogen Activator Inhibitor-1 (PAI-1) (ab66705, Abcam, Waltham, MA, USA). β-actin (sc-47778, Santa Cruz, CA, USA) was used as a reference protein.

Techniques: Expressing, Real-time Polymerase Chain Reaction, TUNEL Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Anti-HMGB1 mAb Therapy Reduces Epidural Hematoma Injury

doi: 10.3390/ijms25115889

Figure Lengend Snippet: Expression of AQP4, PAR1, PAI-1, and MSR1 after the EDH and effects of anti-HMGB1 treatment. ( A ) The protein levels of AQP4 and PAR-1 in the ipsilateral cerebral cortex (beneath the hematoma) were determined by western blotting 48 h after the EDH. The representative results of western blotting are shown. ( B ) The quantitative results are expressed as mean ± SE of 3–5 rats. The protein levels of AQP4 and PAR-1 in the ipsilateral cerebral cortex (beneath the hematoma) were determined by western blotting 48 h after the EDH. ## p < 0.01, and ### p < 0.001 compared to the sham group. * p < 0.05 compared to the control IgG-treated group. ns means no significant difference. ( C ) The protein levels of PAI-1 and MSR-1 in the ipsilateral cerebral cortex (beneath the hematoma) were determined by western blotting 48 h after the EDH. The representative results of western blotting are shown. ( D ) The quantitative results are expressed as mean ± SE of 3–5 rats. The protein levels of PAI-1 and MSR1 in the ipsilateral cerebral cortex (beneath the hematoma) were determined by western blotting 48 h after the EDH. # p < 0.05, and ### p < 0.001 compared to the sham group. ** p < 0.01, and *** p < 0.01 compared to the control IgG-treated group.

Article Snippet: The membranes were blocked with 5% non-fat dry milk and then incubated overnight at 4 °C with primary antibodies against HMGB1 (ab18256, Abcam, Waltham, MA, USA), Heat Shock Protein 27 (Hsp27) (ab2790, Abcam, Waltham, MA, USA), Hsp40 (ab69402, Abcam, Waltham, MA, USA), Hsp60 (ab46798, Abcam, Waltham, MA, USA), Hsp90 (ab13492, Abcam, Waltham, MA, USA), Peroxiredoxin 5 (Prx5) (ab180587, Abcam, Waltham, MA, USA), Prx6 (ab195045, Abcam, Waltham, MA, USA), Aquaporin-4 (AQP4) (ab9512, Abcam, Waltham, MA, USA), Protease-Activated Receptor 1 (PAR1) (PA5-116040, Invitrogen, Carlsbad, CA, USA), and Plasminogen Activator Inhibitor-1 (PAI-1) (ab66705, Abcam, Waltham, MA, USA). β-actin (sc-47778, Santa Cruz, CA, USA) was used as a reference protein.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Anti-HMGB1 mAb Therapy Reduces Epidural Hematoma Injury

doi: 10.3390/ijms25115889

Figure Lengend Snippet: Effects of the anti-HMGB1 mAb on stimulant-induced HMGB1 translocation in neuroblastoma cells in culture. ( A ) SH-SY5Y neuroblastoma cells were stimulated with hemin (1 mM) for 24 h, and the translocation of HMGB1 was observed immunocytochemically. The anti-HMGB1 mAb (10 μg/mL) or anti-KLH mAb (control) was added to the media immediately before the start of incubation. DAPI was used for nuclear staining. Scale bar: 20 μm. ( B ) IL-6 and IL-8 levels in the media stimulated with hemin (0.6 mM) were determined 24 h after stimulation in the presence (10 μg/mL) or absence of the anti-HMGB1 mAb. The results are expressed as mean ± SE of triplicate experiments. * p < 0.05 and ** p < 0.01 compared to the non-stimulated group or to the control IgG-treated group, as shown by the lines in the figure. ( C ) SH-SY5Y neuroblastoma cells were stimulated with FeCl 2 (0.6 mM) for 24 h in the presence or absence of the anti-HMGB1 mAb (10 μg/mL), and the translocation of HMGB1 was observed immunocytochemically. Scale bar: 20 μm. ( D ) SH-SY5Y neuroblastoma cells were stimulated with thrombin (5 U/mL) for 24 h in the presence or absence of the anti-HMGB1 mAb (10 μg/mL), and the translocation of HMGB1 was observed immunocytochemically. Scale bar: 20 μm.

Article Snippet: The membranes were blocked with 5% non-fat dry milk and then incubated overnight at 4 °C with primary antibodies against HMGB1 (ab18256, Abcam, Waltham, MA, USA), Heat Shock Protein 27 (Hsp27) (ab2790, Abcam, Waltham, MA, USA), Hsp40 (ab69402, Abcam, Waltham, MA, USA), Hsp60 (ab46798, Abcam, Waltham, MA, USA), Hsp90 (ab13492, Abcam, Waltham, MA, USA), Peroxiredoxin 5 (Prx5) (ab180587, Abcam, Waltham, MA, USA), Prx6 (ab195045, Abcam, Waltham, MA, USA), Aquaporin-4 (AQP4) (ab9512, Abcam, Waltham, MA, USA), Protease-Activated Receptor 1 (PAR1) (PA5-116040, Invitrogen, Carlsbad, CA, USA), and Plasminogen Activator Inhibitor-1 (PAI-1) (ab66705, Abcam, Waltham, MA, USA). β-actin (sc-47778, Santa Cruz, CA, USA) was used as a reference protein.

Techniques: Translocation Assay, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Anti-HMGB1 mAb Therapy Reduces Epidural Hematoma Injury

doi: 10.3390/ijms25115889

Figure Lengend Snippet: Effects of the anti-HMGB1 mAb on TNF-α-induced HMGB1 translocation in vascular endothelial cells in culture. EA.hy 926 vascular endothelial cells were stimulated with TNF (100 ng/mL) in the presence or absence of the anti-HMGB1 mAb (10 μg/mL) for 6 h, and the translocation of HMGB1 was observed immunocytochemically. DAPI was used for nuclear staining.

Article Snippet: The membranes were blocked with 5% non-fat dry milk and then incubated overnight at 4 °C with primary antibodies against HMGB1 (ab18256, Abcam, Waltham, MA, USA), Heat Shock Protein 27 (Hsp27) (ab2790, Abcam, Waltham, MA, USA), Hsp40 (ab69402, Abcam, Waltham, MA, USA), Hsp60 (ab46798, Abcam, Waltham, MA, USA), Hsp90 (ab13492, Abcam, Waltham, MA, USA), Peroxiredoxin 5 (Prx5) (ab180587, Abcam, Waltham, MA, USA), Prx6 (ab195045, Abcam, Waltham, MA, USA), Aquaporin-4 (AQP4) (ab9512, Abcam, Waltham, MA, USA), Protease-Activated Receptor 1 (PAR1) (PA5-116040, Invitrogen, Carlsbad, CA, USA), and Plasminogen Activator Inhibitor-1 (PAI-1) (ab66705, Abcam, Waltham, MA, USA). β-actin (sc-47778, Santa Cruz, CA, USA) was used as a reference protein.

Techniques: Translocation Assay, Staining

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Short Thymic Stromal Lymphopoietin Attenuates Toluene Diisocyanate-induced Airway Inflammation and Inhibits High Mobility Group Box 1-Receptor for Advanced Glycation End Products and Long Thymic Stromal Lymphopoietin Expression.

doi: 10.1093/toxsci/kfx043

Figure Lengend Snippet: FIG. 3. Pulmonary expressions of RAGE, HMGB1 and mouse TSLP. A–C, Representative immunohistochemistry of RAGE, HMGB1 and mouse TSLP in lung (upper panel)

Article Snippet: Samples were treated with H2O2 for 10 min to block endogenous peroxidase, blocked with 5% BSA for 20 min at room temperature and then stained with monoclonal rabbit anti-HMGB1 antibody (ab79823,1:600, Abcam, Cambridge, MA), polyclonal rabbit anti-RAGE antibody (ab37647,1:100, Abcam), rabbit monoclonal to p-STAT3(Y705) (9145P,1:50, CST, Danvers, MA), rabbit monoclonal to p-STAT5 (Y694) (ab32364,1:50, Abcam) and polyclonal rabbit anti-TSLP antibody (ab188766, 1:100, Abcam) respectively and incubated overnight at 4 C. After washing with PBS, slices were incubated with anti-rabbit IgG (Boster, Wuhan, China) for 20 min and then stained with SABC (Boster) for 20 min at room temperature.

Techniques: Immunohistochemistry

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Short Thymic Stromal Lymphopoietin Attenuates Toluene Diisocyanate-induced Airway Inflammation and Inhibits High Mobility Group Box 1-Receptor for Advanced Glycation End Products and Long Thymic Stromal Lymphopoietin Expression.

doi: 10.1093/toxsci/kfx043

Figure Lengend Snippet: FIG. 6. Expressions of human TSLP variants mRNA and HMGB1, RAGE and long TSLP proteins in 16HBE cells. A and B, 16HBE cells were stimulated with TDI-HSA (80lg/ml) or rhHMGB1 (30ng/ml) for 4h. Both TDI-HSA(A) and rhHMGB1(B) upregulated the long TSLP but not short TSLP mRNA production. C, E, G and I, Western blot analysis of

Article Snippet: Samples were treated with H2O2 for 10 min to block endogenous peroxidase, blocked with 5% BSA for 20 min at room temperature and then stained with monoclonal rabbit anti-HMGB1 antibody (ab79823,1:600, Abcam, Cambridge, MA), polyclonal rabbit anti-RAGE antibody (ab37647,1:100, Abcam), rabbit monoclonal to p-STAT3(Y705) (9145P,1:50, CST, Danvers, MA), rabbit monoclonal to p-STAT5 (Y694) (ab32364,1:50, Abcam) and polyclonal rabbit anti-TSLP antibody (ab188766, 1:100, Abcam) respectively and incubated overnight at 4 C. After washing with PBS, slices were incubated with anti-rabbit IgG (Boster, Wuhan, China) for 20 min and then stained with SABC (Boster) for 20 min at room temperature.

Techniques: Western Blot

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Short Thymic Stromal Lymphopoietin Attenuates Toluene Diisocyanate-induced Airway Inflammation and Inhibits High Mobility Group Box 1-Receptor for Advanced Glycation End Products and Long Thymic Stromal Lymphopoietin Expression.

doi: 10.1093/toxsci/kfx043

Figure Lengend Snippet: FIG. 8. Effects of short TSLP on RAGE and long TSLP proteins expression, HMGB1 and RAGE localization and signals activation in 16HBE cells. Western blot and densito-

Article Snippet: Samples were treated with H2O2 for 10 min to block endogenous peroxidase, blocked with 5% BSA for 20 min at room temperature and then stained with monoclonal rabbit anti-HMGB1 antibody (ab79823,1:600, Abcam, Cambridge, MA), polyclonal rabbit anti-RAGE antibody (ab37647,1:100, Abcam), rabbit monoclonal to p-STAT3(Y705) (9145P,1:50, CST, Danvers, MA), rabbit monoclonal to p-STAT5 (Y694) (ab32364,1:50, Abcam) and polyclonal rabbit anti-TSLP antibody (ab188766, 1:100, Abcam) respectively and incubated overnight at 4 C. After washing with PBS, slices were incubated with anti-rabbit IgG (Boster, Wuhan, China) for 20 min and then stained with SABC (Boster) for 20 min at room temperature.

Techniques: Expressing, Activation Assay, Western Blot

Journal: Oncotarget

Article Title: Stimulation of dendritic cells by DAMPs in ALA-PDT treated SCC tumor cells

doi:

Figure Lengend Snippet: Tumor tissue was collected 1, 3, 6, 9, and 12 h after treatment, stained and observed under different magnifications: for HSP70 at 100× (upper panel) and for HMGB1 and CRT at 400× (middle and lower panels, respectively). The expressions of all three DAMPs were positively increased between 3 to 9 h after ALA-PDT, reaching their peak values at 6 h.

Article Snippet: Rabbit polyclonal anti-HSP70 and mouse monoclonal anti-HMGB1 (Cell Signaling Technology, USA), rabbit polyclonal anti-calreticulin (abcam, USA), mouse monoclonal anti-GAPDH and mouse monoclonal anti-FAS (Santa Cruz, USA) were used.

Techniques: Staining

Journal: Oncotarget

Article Title: Stimulation of dendritic cells by DAMPs in ALA-PDT treated SCC tumor cells

doi:

Figure Lengend Snippet: A. Expression of intracellular CRT. PECA cells were treated by ALA-PDT with different doses (0.5J/cm 2 , 1J/cm 2 , 2J/cm 2 ), and CRT expression was analyzed by western blot at 1 h or 6 h after treatment. The highest expression of CRT was observed under the treatment with the light dose of 0.5J/cm 2 . Intracellular expressions of CRT B. HMGB1 C. and HSP70 D. in PECA cells at different time points (0 h to 9 h) after treatment with a light dose of 0.5J/cm 2 . The expressions of HSP70, HMGB1, and CRT reached their peak values between 3 to 6 h.

Article Snippet: Rabbit polyclonal anti-HSP70 and mouse monoclonal anti-HMGB1 (Cell Signaling Technology, USA), rabbit polyclonal anti-calreticulin (abcam, USA), mouse monoclonal anti-GAPDH and mouse monoclonal anti-FAS (Santa Cruz, USA) were used.

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: Stimulation of dendritic cells by DAMPs in ALA-PDT treated SCC tumor cells

doi:

Figure Lengend Snippet: PDT-stimulated release of HMGB1 from PECA cells at different time points (1 h to 12 h) at 0.5J/cm 2 A. 1J/cm 2 B. and 2J/cm 2 C. using ELISA assay. D. The peak values of HMGB1 6 h after treatment. Statistical analysis was performed by t -test; * p < 0.05, ** p < 0.01 and *** p < 0.001; ns = not statistically significant. Means ± SD are shown from 3 independent experiments.

Article Snippet: Rabbit polyclonal anti-HSP70 and mouse monoclonal anti-HMGB1 (Cell Signaling Technology, USA), rabbit polyclonal anti-calreticulin (abcam, USA), mouse monoclonal anti-GAPDH and mouse monoclonal anti-FAS (Santa Cruz, USA) were used.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Stimulation of dendritic cells by DAMPs in ALA-PDT treated SCC tumor cells

doi:

Figure Lengend Snippet: PECA cells were collected 6 h after ALA-PDT treatment (0.5 mM ALA, 0.5 J/cm 2 ), then co-incubated with imDCs for another 24 h. The stimulation of DCs was at the similar level as that of LPS (3 rd and 4 th panels). Blocking HSP70, HMGB1 and CRT individually reduced the expression of MHC-II, CD80, and CD86 on DCs (5, 6, and 7 th panels); however, blocking all three DAMPs resulted in the least DC activation (8 th panel). PECA-DCs represents untreated PECA cells were collected and incubated with imDCs for 24 h, and PDT-DCs represents DCs stimulated with PDT-treated PECA cells. Three represents HSP70, HMGB1 and CRT, and (−) represents blocking.

Article Snippet: Rabbit polyclonal anti-HSP70 and mouse monoclonal anti-HMGB1 (Cell Signaling Technology, USA), rabbit polyclonal anti-calreticulin (abcam, USA), mouse monoclonal anti-GAPDH and mouse monoclonal anti-FAS (Santa Cruz, USA) were used.

Techniques: Incubation, Blocking Assay, Expressing, Activation Assay

Journal: Oncotarget

Article Title: Stimulation of dendritic cells by DAMPs in ALA-PDT treated SCC tumor cells

doi:

Figure Lengend Snippet: Secretion of IFN-γ A. and IL-12 B. from DCs after incubation with ALA-PDT-treated PECA cells was quantified in cell culture supernatants using ELISA assay, with or without HSP70, HMGB1 or CRT neutralizing antibodies in the co-incubation medium. Unstimulated, immature DCs were used for negative control and DCs incubated with LPS were used for positive control. ALA-PDT-treated PECA cells induced the secretion of IFN-γ and IL-12 markedly higher than that induced by untreated PECA cells ( p < 0.05). When HSP70, HMGB1 and CRT were blocked individually, or in combination, IFN-γ secreted from DCs was markedly reduced ( p < 0.05). When all of three antibodies were included in the co-incubation medium, IL-12 secreted from DCs significantly decreased when compared with PDT-DCs ( p < 0.05). Statistical analysis was performed by t -test; * p < 0.05 and ** p < 0.01 with respect to the PDT-DCs (unless otherwise specified); ns = not statistically significant. Means ± SD are shown ( n = 5 from 3 independent experiments.)

Article Snippet: Rabbit polyclonal anti-HSP70 and mouse monoclonal anti-HMGB1 (Cell Signaling Technology, USA), rabbit polyclonal anti-calreticulin (abcam, USA), mouse monoclonal anti-GAPDH and mouse monoclonal anti-FAS (Santa Cruz, USA) were used.

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Negative Control, Positive Control

Journal: Journal of Innate Immunity

Article Title: Limited Role of the Receptor for Advanced Glycation End Products during Streptococcus pneumoniae Bacteremia

doi: 10.1159/000348739

Figure Lengend Snippet: Expression of RAGE in mouse organs and of its ligand HMGB1 in plasma after systemic pneumococcal infection. a Representative picture of a lung sample from a normal, uninfected Wt mouse, displaying extensive RAGE staining. b Lung sample from a Wt mouse 48 h after intravenous injection of 5 × 105 CFU of S. pneumoniae. c Absence of RAGE positivity in the lung of a rage−/− mouse. Scale bar = 200 μm. RAGE concentrations in lungs (d), spleens (e) and livers (f) from naïve Wt mice and 24 and 48 h after intravenous injection of 5 × 105 CFU of S. pneumoniae (5-8 mice per group at each time point). * p < 0.05 versus naïve mice (0 h). g Densitometric analysis of HMGB1 blot expressed as a percentage of the mean density measured in naïve control plasma samples. HMGB1 detection was enhanced at 24 and 48 h compared to naïve control samples (0 h; n = 3 per time point). Bars represent mean ± standard error of the mean.

Article Snippet: Following blocking with 5% nonfat dry milk proteins (Protifar from Nutricia, Zoetermeer, The Netherlands) in 0.1% Tween phosphate-buffered saline (PBS-T), membranes were washed and incubated overnight in 1 µg/ml primary rabbit anti-HMGB1 polyclonal antibody (ab18256, Abcam, Cambridge, UK) in 1% nonfat dry milk proteins in PBS-T at 4°C.

Techniques: Expressing, Infection, Staining, Injection