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: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Stimulatory effects of advanced glycation endproducts (AGEs) on fibronectin matrix assembly

doi: 10.1016/j.matbio.2016.07.003

Figure Lengend Snippet: Mesangial cells were grown for 2 days on tissue culture plastic + AGE-BSA or BSA (20 µg/mL) as in Figure 5 legend. 10 µg/mL rat plasma FN and 15 µg/ml RAGE function-blocking antibody were added for the final 24 hours. (A) Phase and fluorescence images were captured as described in Figure 5 legend. Average FN fluorescence intensity was decreased 1.16-fold to 0.86 ± range (n = 2) in the presence of RAGE function-blocking antibody with AGE-BSA compared to AGE-BSA treatment alone. Scale bar is 200 µm. (B) The DOC-insoluble fraction was separated by SDS-PAGE and immunoblotted with IC3 anti-rat FN monoclonal. Relative densitometry values are the mean of two experiments ± range normalized to GAPDH from the DOC soluble fraction. Blot is representative of two independent experiments.

Article Snippet: 15 μg/ml RAGE function blocking antibody (AF1179, R&D Systems, Minneapolis, MN) or lysyl oxidase (LOX) antibody (ab31238, Abcam, Cambridge, MA) was added for final 24 hours of 48 hour time-course where indicated.

Techniques: Clinical Proteomics, Blocking Assay, Fluorescence, SDS Page