Journal: Biochimica et biophysica acta

Article Title: RAGE overexpression confers a metastatic phenotype to the WM115 human primary melanoma cell line.

doi: 10.1016/j.bbadis.2014.02.013

Figure Lengend Snippet: Fig. 1. Q2 A) Levels of RAGE in RAGE transfected WM115 cells as determined by ELISA. Levels are expressed in pg RAGE protein per mg of total protein. WM115-RAGE and WM115-RAGE-I expressed 94 fold and 7 fold higher RAGE protein than the MOCK control cells, respectively. B) Binding of the anti-RAGE antibody MAB1145 to WM115-RAGE (filled circles) and WM115- MOCK (filled squares) measured by flow cytometry. The binding curve of MAB1145 to WM115-RAGE was fitted using a 1:1 binding model and showed an affinity of 1.5 (±0.3) nM. RAGE overexpressed in the melanoma cells is properly processed and translocated to the cell-surface, as demonstrated by their recognition by specific antibodies. The experiment was performed in triplicate and the standard deviation is indicated. C–F) Morphology of WM115-MOCK (C), WM115-RAGE-I (D), WM115-RAGE (E) and WM266-MOCK (F) by bright field microscopy. G–H) Differences in morphology between WM115-MOCK (G) and WM115-RAGE (H) transfected cells, as shown by actin staining. Actin was stained with PE conjugated phalloidin and the nuclei were stained with Hoechst 33342. (20× magnification).

Article Snippet: RAGE protein levels in the cell extracts were determined using the Quantikine human RAGE Immunoassay kit (R&D Systems) according to the manufacturer's procedure, and expressed in picogram of RAGE per mg of total protein.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Control, Binding Assay, Cytometry, Standard Deviation, Microscopy, Staining

Journal: Molecules and Cells

Article Title: Characterization of αX I-Domain Binding to Receptors for Advanced Glycation End Products (RAGE)

doi: 10.14348/molcells.2017.0021

Figure Lengend Snippet: Binding of αX and αM I-domains to RAGE and the V-domain of RAGE. (A) A schematic representation of recombinant RAGE and RAGE derived soluble domains. All soluble proteins are fused with a His-tag for purification and detection. (B) SDS-PAGE analysis of purified sRAGE, sRAGEC1/2 and sRAGEV. (C) SPR sensorgram of sRAGE and RAGE-derived soluble domains binding to immobilized GST-αX-I. RAGE-derived proteins (1 μM) were injected to flow over immobilized GST-αX-I on a CM5 sensor chip (1800 RU). (D) Binding of sRAGEV and sRAGEC1/2 to GST-αX-I on microtiter plates. sRAGEV and sRAGEC1/2 (0.5 μM or 1.0 μM) were loaded on microtiter plates coated with GST-αX-I. Data are means ± S. E. (n = 3). (E, F) Binding of the I-domains to the sRAGE (E) and sRAGEV (F) on microtiter plates. GST and αX and αM I-domains (0.5 μM or 1.0 μM) were loaded on microtiter plates coated with sRAGE and sRAGEV. Data are means ± S. E. (n = 3).

Article Snippet: Recombinant RAGE fused with the human IgG Fc region produced from mammalian cells was purchased from R&D Systems (USA).

Techniques: Binding Assay, Recombinant, Derivative Assay, Purification, SDS Page, Injection

Journal: iScience

Article Title: Direct interaction of HMGB1 with SARS-CoV-2 facilitates its infection via RAGE-dependent endocytosis

doi: 10.1016/j.isci.2025.113063

Figure Lengend Snippet: HMGB1 induces SARS-CoV-2 infection in an ACE2-independent RAGE-dependent manner (A) Western blot analysis of receptors responsible for SARS-CoV-2 infection and HMGB1 binding. S.E., short exposure; L.E., long exposure. (B) Flow cytometry analysis of ectodomain ACE2 in A549 and NCI-H1975 cells used in this study. Vero E6 cells were used as control. (C) A549 cells were transfected with shRNA-ACE2 for 48 h prior to infection with 1 MOI SARS-CoV-2, which was preincubated with HMGB1 for 1 h at 37°C, cultured further for 3 h, and subjected to western blotting. (D and E) A549 cells were infected for 1 h with 1 MOI SARS-CoV-2, which was preincubated with HMGB1 for 1 h, in the presence of 40 μg/mL sRAGE at 37°C as indicated. Cells were harvested at 3 hpi and subjected to western blotting (D) n = 3, and qRT-PCR for viral RNA measurement (E) n = 3. (F) NCI-H1975 cells were infected with SARS-CoV-2 preincubated with 20 μg/mL HMGB1 in the presence of azeliragon. Cells were harvested at 3 hpi and subjected to western blotting. (G) A549 cells were infected with 5 MOI SARS-CoV-2, which was treated as above to observe NP. Representative confocal images are shown. The percentage of infected cells and NP intensity were measured by counting at least 700 visible cells. n = 4. (H) Cycloheximide pretreated NCI-H1975 cells were infected with 5 MOI SARS-CoV-2 (preincubated with HMGB1). Cells were stained for NP before permeabilization (green; external) and post-permeabilization (red; external and internal). Representative images and their magnifications are shown. The percentage of intracellular spots was measured by counting at least 200 visible cells. n = 4. (I and J) SARS2pp was preincubated with or without HMGB1 in the presence of sRAGE before transduction in NCI-H1975 cells. NanoLuc luciferase activity was measured 72 h post-transduction. n = 3 Scale bars represent 5 μm. Data are presented as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant, using one-way ANOVA with Tukey’s multiple comparison test and Student’s unpaired t-test. HMGB1, high-mobility group box 1; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; MOI, multiplicity of infection; qRT-PCR, quantitative reverse transcription polymerase chain reaction; NP, nucleocapsid protein; SEM, standard error of the mean; ANOVA, analysis of variance; hpi, hours post-infection; RAGE, receptor for advanced glycation end-products; ACE2, angiotensin-converting enzyme 2; SARS2pp, SARS-CoV-2 spike protein (S)-pseudotyped retrovirus.

Article Snippet: 40 μg/mL sRAGE (11629-HCCH, Sino Biological, Oklahoma City, OK, USA), azeliragon (S6415, Selleckchem, Houston, TX, USA), dynasore (D7693, Sigma-Aldrich, St. Louis, MO, USA) and chloroquine (C6628, Sigma-Aldrich) were pretreated for 2 h at 37°C, prior to the infection procedure.

Techniques: Infection, Western Blot, Binding Assay, Flow Cytometry, Control, Transfection, shRNA, Cell Culture, Quantitative RT-PCR, Staining, Transduction, Luciferase, Activity Assay, Comparison, Reverse Transcription, Polymerase Chain Reaction