rage blocking antibody  (R&D Systems)

Journal: Molecular Medicine Reports

Article Title: S100A8 and S100A9 promote endothelial cell activation through the RAGE-mediated mammalian target of rapamycin complex 2 pathway

doi: 10.3892/mmr.2020.11595

Figure Lengend Snippet: S100A8/9-induced HUVEC activation is RAGE-dependent. HUVECs were pretreated with 10 µg/ml RAGE blocking antibody or 100 nM rapamycin for 1 h prior to treating with S100A8/9 for 48 h. Total protein was harvested and subjected to western blotting. (A) RAGE antibody pretreatment blocked the PI3K/Akt/mTOR pathway, whereas rapamycin only reduced mTOR phosphorylation. (B) RAGE antibody pretreatment blocked Rictor and PKCα, whereas rapamycin made no difference. (C) The change in Rictor mRNA expression was measured by reverse transcription-quantitative PCR. Data and error bars represent the mean ± SEM (n=3). *P<0.05, **P<0.01 vs. control group; # P<0.05, ## P<0.01 vs. S100A8/9 group. RAGE, receptor for advanced glycation end products; S100A, S100 calcium binding protein A; Rictor, rapamycin-insensitive companion of mTOR; PKCα, protein kinase Cα; p-, phosphorylated; t-, total.

Article Snippet: HUVECs were pretreated with 10 μg/ml RAGE blocking antibody (cat. no. MAB11451; R&D Systems) or 100 nM rapamycin for 1 h prior to treating with S100A8/9 for 48 h at 37°C with 5% CO 2 .

Techniques: Activation Assay, Blocking Assay, Western Blot, Phospho-proteomics, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Binding Assay

Journal: Molecular Medicine Reports

Article Title: S100A8 and S100A9 promote endothelial cell activation through the RAGE-mediated mammalian target of rapamycin complex 2 pathway

doi: 10.3892/mmr.2020.11595

Figure Lengend Snippet: Effects of RAGE blockade on S100A8/9 stimulation of the PI3K/Akt/mTOR and mTORC2 signaling pathways. (A) Human umbilical vein endothelial cells transfected with si-RAGE, si-Rictor or control, as indicated, were subjected to serum starvation for 24 h. Total protein was harvested and subjected to western blotting. (B) Total RNA was harvested and subjected to reverse transcription-quantitative PCR. (C) Viability was assessed by Cell Counting Kit-8 assays. The relative cell viability ratios are expressed as a percentage of the 48 h control group. (D) RAGE and Rictor knockdown by siRNA can partially reverse the S100A8/9-induced increase in cell migration. (E) RAGE and Rictor knockdown by siRNA can partially prevent the S100A8/9-induced angiogenesis. Data and error bars represent the mean ± SEM (n=3). *P<0.05, **P<0.01 vs. control group; ## P<0.01 vs. S100A8/9 group. RAGE, receptor for advanced glycation end products; S100A, S100 calcium binding protein A; Rictor, rapamycin-insensitive companion of mTOR; siRNA, small interfering RNA; NC, negative control.

Article Snippet: HUVECs were pretreated with 10 μg/ml RAGE blocking antibody (cat. no. MAB11451; R&D Systems) or 100 nM rapamycin for 1 h prior to treating with S100A8/9 for 48 h at 37°C with 5% CO 2 .

Techniques: Protein-Protein interactions, Transfection, Control, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Knockdown, Migration, Binding Assay, Small Interfering RNA, Negative Control

Journal: Clinical and Experimental Immunology

Article Title: High fructose‐induced metabolic changes enhance inflammation in human dendritic cells

doi: 10.1111/cei.13299

Figure Lengend Snippet: Acute exposure (24 h) of dendritic cells (DCs) to high fructose induces advanced glycation end products (AGE) formation and activates the AGE–receptor for advanced glycation end product (RAGE) pathway. DCs cultured in media supplemented with 15 mM glucose/fructose for 24 h or normal media were investigated for the effect on AGE–RAGE pathway. (a) Immunocytochemistry of AGE in DCs ± amino guanidine (AGE blocker); (b) quantification of AGE accumulation in supernatants using enzyme‐linked immunosorbent assay (ELISA). Data are mean ± s.e. of five donors. (c) Quantification of AGE in the DC supernatants by ELISA ± amino guanidine. Data are mean ± s.e. of three donors. (d) Immunocytochemistry of RAGE and (e) RAGE surface expression using flow cytometry. (a,c) Representative of three such experiments with different donors.

Article Snippet: Immunocytochemistry of AGE, RAGE and nuclear factor kappa B (NF‐kB) DCs were seeded onto glass coverslips in 24‐well plates (0·5 × 10 6 cells/ml) in RPMI and treated with 15 mM glucose/fructose for 24 h with or without 1 mM AMG or blocking antibody against RAGE (R&D Systems; clone 176902, 10 µg/ml) added 30 min prior to glucose/fructose treatment.

Techniques: Cell Culture, Immunocytochemistry, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

Journal: Clinical and Experimental Immunology

Article Title: High fructose‐induced metabolic changes enhance inflammation in human dendritic cells

doi: 10.1111/cei.13299

Figure Lengend Snippet: Enhanced activation of nuclear factor kappa b (NF)‐κB signaling pathway in fructose exposed dendritic cells (DCs) and specific role of fructose–advanced glycation end products (AGE). DCs cultured in 15 mM glucose/fructose were investigated for the effect on the NF‐κB pathway, the downstream effector of AGE–receptor for advanced glycation end product (RAGE) pathway activation by assaying (a). Phospho‐Iκbα by flow cytometry; (b) immunocytochemistry of p65 localization in the nucleus; (c) bar graphs depict the effect of AGE–RAGE pathway inhibition on cytokine production by DCs. Glucose–AGE (G–AGE) and fructose–AGE (F–AGE) were prepared in vitro after incubation of the sugars with bovine serum albumin for 6–8 weeks. The specific effect of these AGEs on DCs was evaluated after stimulation for 24 h. (d) Quantification of AGE formation using fluorescence; (f) Effect of G–AGE and F–AGE on phospho‐Iκbα levels using flow cytometry; (f) bar graphs depict the effect of F‐AGE and G‐AGE and unconjugated BSA on cytokines secretion by DCs at 24 h. Data are mean ± of three experiments with different donors.

Article Snippet: Immunocytochemistry of AGE, RAGE and nuclear factor kappa B (NF‐kB) DCs were seeded onto glass coverslips in 24‐well plates (0·5 × 10 6 cells/ml) in RPMI and treated with 15 mM glucose/fructose for 24 h with or without 1 mM AMG or blocking antibody against RAGE (R&D Systems; clone 176902, 10 µg/ml) added 30 min prior to glucose/fructose treatment.

Techniques: Activation Assay, Cell Culture, Flow Cytometry, Immunocytochemistry, Inhibition, In Vitro, Incubation, Fluorescence