Journal: Frontiers in Cellular Neuroscience

Article Title: Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fncel.2015.00485

Figure Lengend Snippet: Expression of RAGE and its ligands in control and ALS thoracic spinal cord tissue. (A) RAGE expression in control ( A , top), and in ALS tissue ( A , bottom). (B) S100B immunostaining in control tissue ( B , top) and in ALS tissue ( B , bottom). (C) HMGB1 immunostaining in the control tissue ( C , top) and in ALS tissue ( C , bottom). (D) CML immunostaining in control spinal cord ( D , top) and in ALS spinal cord ( D , bottom). (E–G) Quantification of immunostaining intensity revealed that expression of all studied proteins was significantly increased in ALS thoracic spinal cord tissue compared to controls. S100B (E) was increased about 70%, HMGB1 (F) displayed almost threefold increase and CML (G) showed almost double level of increase in immunostaining between ALS and control subjects. Sections are representative of n = 6 control and n = 5 ALS tissue samples per condition. Error bars represent mean ± SEM, ∗ p < 0.05. Scale bar: 50 μm.

Article Snippet: Tissue homogenates (30 μg) were subjected to electrophoresis using 4–12% SDS-PAGE gels and specific protein signals were detected using the following antibodies: mouse anti-β-actin (A1978, 1:4000, Sigma); rabbit anti-RAGE (GTX23611, 1:1000, GeneTex); rabbit anti-S100B (ab52642, 1:5000, Abcam); and rabbit anti-HMGB1 (GTX101277, 1:1000, GeneTex).

Techniques: Expressing, Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fncel.2015.00485

Figure Lengend Snippet: High magnification images of immunostaining for RAGE and its ligands in the thoracic spinal cord. Increased immunostaining pattern on the border of gray (lamina IX) and white matter was observed for (A) RAGE, (B) S100B, (C) HMGB1 and (D) CML in ALS versus control samples. Scale bar: 50 μm.

Article Snippet: Tissue homogenates (30 μg) were subjected to electrophoresis using 4–12% SDS-PAGE gels and specific protein signals were detected using the following antibodies: mouse anti-β-actin (A1978, 1:4000, Sigma); rabbit anti-RAGE (GTX23611, 1:1000, GeneTex); rabbit anti-S100B (ab52642, 1:5000, Abcam); and rabbit anti-HMGB1 (GTX101277, 1:1000, GeneTex).

Techniques: Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fncel.2015.00485

Figure Lengend Snippet: Co-expression of RAGE and RAGE ligands S100B, CML, and HMGB1 is higher in human ALS spinal cord. (A) Triple staining for RAGE (red), S100B (green), CML (blue) revealed increased immunoexpression of these proteins in the ALS spinal cord ( A , right) as compared to controls ( A , left) and a high degree of RAGE/ligand overlapping was observed in ALS samples (merged images). (B) Expression of RAGE (red) and its ligands, S100B (green) and HMGB1 (blue) was highly increased in the ALS ( B , right) spinal cord as compared to controls ( B , left) and a high degree of RAGE/ligand co-expression observed in ALS samples (merged images); control ( n = 6) vs. ALS samples ( n = 5). Scale bar: 100 μm. (C) A schematic diagram showing different regions of spinal cord; for the purpose of the study we examined thoracic motor spinal cord ventral horn lamina IX and surrounding white matter.

Article Snippet: Tissue homogenates (30 μg) were subjected to electrophoresis using 4–12% SDS-PAGE gels and specific protein signals were detected using the following antibodies: mouse anti-β-actin (A1978, 1:4000, Sigma); rabbit anti-RAGE (GTX23611, 1:1000, GeneTex); rabbit anti-S100B (ab52642, 1:5000, Abcam); and rabbit anti-HMGB1 (GTX101277, 1:1000, GeneTex).

Techniques: Expressing, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fncel.2015.00485

Figure Lengend Snippet: High magnification images of white/gray matter showing triple staining for RAGE and its ligands S100B, CML, and HMGB1. Immunostaining for RAGE (red) and its ligands S100B (green) and CML or HMGB1 (blue) revealed low immunoexpression in control tissue ( A and C ) and high immunoexpression in ALS tissue ( B and D ). Sections are representative of n = 6 control and n = 5 ALS tissue samples per condition. Scale bar: 100 μm.

Article Snippet: Tissue homogenates (30 μg) were subjected to electrophoresis using 4–12% SDS-PAGE gels and specific protein signals were detected using the following antibodies: mouse anti-β-actin (A1978, 1:4000, Sigma); rabbit anti-RAGE (GTX23611, 1:1000, GeneTex); rabbit anti-S100B (ab52642, 1:5000, Abcam); and rabbit anti-HMGB1 (GTX101277, 1:1000, GeneTex).

Techniques: Staining, Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fncel.2015.00485

Figure Lengend Snippet: Protein levels of RAGE and its ligands S100B and HMGB1 are higher in human ALS spinal cord. Western blot analysis of RAGE (A) , RAGE ligands S100B (B) and HMGB1 (C) in control and ALS spinal cord tissue. The original blots were stripped followed by incubation with the other antigens under study. Signal for test antigen was then normalized to β-actin and the relative band densities were reported. n = 3 subjects/group. Error bars represent mean ± SEM, ∗ p < 0.05.

Article Snippet: Tissue homogenates (30 μg) were subjected to electrophoresis using 4–12% SDS-PAGE gels and specific protein signals were detected using the following antibodies: mouse anti-β-actin (A1978, 1:4000, Sigma); rabbit anti-RAGE (GTX23611, 1:1000, GeneTex); rabbit anti-S100B (ab52642, 1:5000, Abcam); and rabbit anti-HMGB1 (GTX101277, 1:1000, GeneTex).

Techniques: Western Blot, Incubation

Journal: Frontiers in Cellular Neuroscience

Article Title: Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fncel.2015.00485

Figure Lengend Snippet: A proposed mechanism of RAGE action in ALS spinal cord. We propose that during pathological processes in ALS, neuronal and microglial RAGE becomes activated by RAGE ligands such as AGEs, S100B, and HMGB1. Once activated, RAGE triggers a cascade of metabolic changes, contributing to the release of reactive oxygen species (ROS) and inflammatory cytokines, subsequently resulting in altered protein structures and misfolded protein accumulation, impaired mitochondrial function and growing energy deficits ultimately leading to neuronal dysfunction and apoptosis.

Article Snippet: Tissue homogenates (30 μg) were subjected to electrophoresis using 4–12% SDS-PAGE gels and specific protein signals were detected using the following antibodies: mouse anti-β-actin (A1978, 1:4000, Sigma); rabbit anti-RAGE (GTX23611, 1:1000, GeneTex); rabbit anti-S100B (ab52642, 1:5000, Abcam); and rabbit anti-HMGB1 (GTX101277, 1:1000, GeneTex).

Techniques:

Journal: Journal of Pain Research

Article Title: Melatonin MT2 receptor agonist IIK-7 produces antinociception by modulation of ROS and suppression of spinal microglial activation in neuropathic pain rats

doi: 10.2147/JPR.S214671

Figure Lengend Snippet: Continuous infusion of IIK-7 attenuated PSNT-induced activation of HMGB-1, pERK1/2, iNOS, STAT3 and caspase-3 in vivo. Representative immunoblots are HMGB-1, P44/42 MAPK, iNOS, STAT 3 and casp-3 from the left dorsal quadrant portion of the lumbar spinal cord lysate of sham surgery or PSNT rats continuously infused with either vehicle or IIK-7 for 7 days using an osmotic pump. Abbreviations: IIK-7, N-Butanoyl 2-(9-methoxy-6H-iso-indolo[2,1-a]indol-11-yl)-ethan-amine; P44/42 MAPK, P44/42 mitogen-activated protein kinase; iNOS, Inducible nitric oxide synthase; STAT3, Signal transducer and activator of transcription 3; CASP-3, caspase3; HMGB-1, High mobility group box 1.

Article Snippet: Primary antibodies against HMGB-1 (Wuhan Fine Biotech, Wuhan, China, Cat # FNab03924), p44/42 MAPK (Erk1/2) (Cell Signaling Technology, Beverly, MA, USA, Cat # 9102), STAT3 (Milliopre, USA Cat# 06–596), iNOS (Thermo Fischer Scientific, MA, USA, Cat# PA3-030A), casp-3 (Imgenex, Novus Biologicals, Littleton, CO, USA, Cat # 31A1067), β-actin (Sigma-Aldrich, St Louis, MO, USA) were incubated at 4°C overnight followed by the wash with TBST.

Techniques: Activation Assay, In Vivo, Western Blot

Journal: Yonsei Medical Journal

Article Title: Development of Advanced Atherosclerotic Plaque by Injection of Inflammatory Proteins in a Rabbit Iliac Artery Model

doi: 10.3349/ymj.2016.57.5.1095

Figure Lengend Snippet: Morphologic changes of rabbit iliac artery analysis according to immunohistochemistry. Tissue staining of induced atherosclerosis iliac artery plaques in four groups of rabbits. The inflammation content of the plaques is detected by immunohistochemical staining for RAGE, HMGB1, and TNF-α; the macrophage content of the plaques is detected by immunohistochemical staining for the anti-rabbit macrophage clone RAM11. Representative examples of immunohistochemical stain of RAGE (A-D), HMGB1 (E-H), TNF-α (I-L), and RAM11 (M-P) in the rabbit iliac (amplification ×100) (scale bars=100 µm). Lesions of macrophage were markedly less for saline group and oil group, which showed significant differences in the total percent area in comparison to the HMGB1 group and TNF-α group on RAM11 immunohistochemical staining. * p <0.05, compared with the Saline group, † p <0.05, compared with the Oil group. RAGE, receptor for advanced glycation end products; HMGB1, high-mobility group protein B1; TNF-α, tumor necrosis factor-α.

Article Snippet: Membranes were blocked by a 5% skim milk (Noble Bio, Hwaseong, Korea) dilution in Tris-Buffered Saline and Tween 20 (TBS-T) at room temperature for 1 hour, and were subsequently washed three times in TBS-T. Membranes were incubated in TBS-T with primary antibodies against HMGB1, TNF-α, and RAGE (all 1:1000 in 5% BSA, Biosesang, Seongnam, Korea) overnight at 4°C.

Techniques: Immunohistochemistry, Staining, Immunohistochemical staining, Amplification, Saline, Comparison

Journal: Yonsei Medical Journal

Article Title: Development of Advanced Atherosclerotic Plaque by Injection of Inflammatory Proteins in a Rabbit Iliac Artery Model

doi: 10.3349/ymj.2016.57.5.1095

Figure Lengend Snippet: The relative mRNA levels in induced atherosclerosis of rabbit iliac artery. Reverse transcription (RT)-PCR analysis of RAGE, HMGB1, and TNF-α mRNA expression in iliac arteries from four groups of rabbits A. Representative data showing the mRNA expressions of RAGE, HMGB1, and TNF-α in iliac arteries from four groups of rabbits (normalized with GAPDH as the house keeping gene) B, C, and D. The data in the bar graph are quantified ratios of the signal for RAGE, HMGB1, and TNF-α to that for GAPDH set at fold increase. Data are presented as the mean±SEM. * p <0.05, compared with the Saline group, † p <0.05, compared with the Oil group. RAGE, receptor for advanced glycation end products; HMGB1, high-mobility group protein B1; TNF-α, tumor necrosis factor-α; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SEM, standard error of the mean.

Article Snippet: Membranes were blocked by a 5% skim milk (Noble Bio, Hwaseong, Korea) dilution in Tris-Buffered Saline and Tween 20 (TBS-T) at room temperature for 1 hour, and were subsequently washed three times in TBS-T. Membranes were incubated in TBS-T with primary antibodies against HMGB1, TNF-α, and RAGE (all 1:1000 in 5% BSA, Biosesang, Seongnam, Korea) overnight at 4°C.

Techniques: Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Expressing, Saline

Journal: Yonsei Medical Journal

Article Title: Development of Advanced Atherosclerotic Plaque by Injection of Inflammatory Proteins in a Rabbit Iliac Artery Model

doi: 10.3349/ymj.2016.57.5.1095

Figure Lengend Snippet: The relative protein levels in induced atherosclerosis of rabbit iliac artery. Western blot analysis of RAGE, HMGB1 and TNF-α mRNA expressions in iliac artery from four groups of rabbits A. Representative data showing the protein expressions of RAGE, HMGB1 and TNF-α levels in iliac arteries from four groups of rabbits (normalized with GAPDH as the house keeping gene) B, C, and D. The data in the bar graph are quantified ratios of the signal for RAGE, HMGB1, and TNF-α to that for GAPDH set at fold increase. Data were presented as the mean±SEM. * p <0.05, compared with the Saline group, † p <0.05, compared with the Oil group, ‡ p <0.05, compared with the TNF-α group. RAGE, receptor for advanced glycation end products; HMGB1, high-mobility group protein B1; TNF-α, tumor necrosis factor-α; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SEM, standard error of the mean.

Article Snippet: Membranes were blocked by a 5% skim milk (Noble Bio, Hwaseong, Korea) dilution in Tris-Buffered Saline and Tween 20 (TBS-T) at room temperature for 1 hour, and were subsequently washed three times in TBS-T. Membranes were incubated in TBS-T with primary antibodies against HMGB1, TNF-α, and RAGE (all 1:1000 in 5% BSA, Biosesang, Seongnam, Korea) overnight at 4°C.

Techniques: Western Blot, Saline