Human/Mouse/Rat RAGE/AGER Antibody

Detection of Human, Mouse, and Rat RAGE by Western Blot. Western blot shows lysates of human, mouse, and rat lung tissue. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human/Mouse/Rat RAGE Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1145) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). Specific bands were detected for RAGE at approximately 45-55 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human RAGE by Western Blot. Western blot shows lysates of human lung tissue and HEK001 human epidermal keratinocyte cell line. PVDF Membrane was probed with 1 µg/mL of Goat Anti-Human/Mouse/Rat RAGE Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1145) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). Specific bands were detected for RAGE at approximately 43 kDa under non-reducing (NR) conditions and 50 kDa under reducing (R) conditions (as indicated). This experiment was conducted using Immunoblot Buffer Group 5.

RAGE in Human Alzheimer's Disease Brain. RAGE was detected in immersion fixed paraffin-embedded sections of human Alzheimer's disease brain (cerebellum) using 15 µg/mL Goat Anti-Human/Mouse/Rat RAGE Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1145) overnight at 4 °C. Tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

RAGE in Human Lung. RAGE was detected in immersion fixed paraffin-embedded sections of human lung using Goat Anti-Human/Mouse/Rat RAGE Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1145) at 0.5 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC004). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cell membranes. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Detection of Mouse AGER by Western Blot RAGE expression mediates cell aggregation through homophilic interactions.(A) Western blot analysis on control preB 300.19 cells (preB/pCAGS) or expressing FL-RAGE (preB/FL-RAGE) using alpha RAGE N-term1 antibody. Forty µg of cell lysate were loaded and detection of GAPDH was used as loading control. (B) Cell aggregation assay. preB/pCAGS or preB/FL-RAGE cells were cultured for 2, 6 or 24 hours before being photographed at 20× magnification in phase contrast (left panel). Bar corresponds to 50 µm. Quantification of cell aggregates area (right panel). Results are displayed as means±SEM (*, P<0.05; **, P<0.01; ***, P<0.0001). (C) Mixed cell aggregation assay. Red preB (Red-preB) or preB/FL-RAGE (expressing GFP; preB/FL-RAGE-GFP) cells were grown as single cell line suspension or mixed in equal number for 24 hours. Red-preB/FL-RAGE-GFP were mixed with preB/FL-RAGE-GFP for the same time. Bar corresponds to 50 µm. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24475194), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Rat AGER by Western Blot RAGE expression enhances cell-matrix adhesion.(A) Western blot analysis on rat lung lysate and R3/1 cells using three different antibodies recognizing RAGE extracellular (anti-RAGE N-term1 and anti-RAGE N-term2) or intracellular (anti-RAGE C-term) domains. Asterisk (*) indicates nonspecific bands. Forty µg of cell lysate were loaded and detection of GAPDH was used as loading control. (B) Western blot analysis on R3/1-pLXSN and R3/1-FL-RAGE cells using anti-RAGE N-term1 antibody. Forty µg of cell lysate were loaded and detection of GAPDH was used as loading control. (C, D) Cell-matrix adhesion assay. Adhesion of R3/1-pLXSN or R3/1-FL-RAGE cells onto culture dishes coated with 10 µg/ml ECM proteins. Adhesion to PBS, collagen I (Coll I), Fibronectin (FN), or Laminin (Lam) was assayed for 15 minutes (C) or 45 minutes (D). One representative experiment out of three is shown. Results from triplicate wells are displayed as means±SEM (ns, not significant; *, P<0.05; **, P<0.01). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24475194), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human AGER by Immunohistochemistry Differential expression of RAGE and S100A8/A9 on protein level in whole skin.The expression level was analysed by immunochistochemistry using specific anti human S100A8/A9 and anti human RAGE antibodies. The expression was tested in 5 patient samples per group (IC and OTR with in situ or invasive SCC). a: Expression of RAGE and S100A8/A9 in IC and OTR patients with in situ SCC in comparison to normal skin. b: Expression of RAGE and S100A8/A9 in IC and OTR patient with invasive SCC in comparison to normal skin. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0120971), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Rat AGER by Western Blot RAGE expression enhances cell-matrix adhesion.(A) Western blot analysis on rat lung lysate and R3/1 cells using three different antibodies recognizing RAGE extracellular (anti-RAGE N-term1 and anti-RAGE N-term2) or intracellular (anti-RAGE C-term) domains. Asterisk (*) indicates nonspecific bands. Forty µg of cell lysate were loaded and detection of GAPDH was used as loading control. (B) Western blot analysis on R3/1-pLXSN and R3/1-FL-RAGE cells using anti-RAGE N-term1 antibody. Forty µg of cell lysate were loaded and detection of GAPDH was used as loading control. (C, D) Cell-matrix adhesion assay. Adhesion of R3/1-pLXSN or R3/1-FL-RAGE cells onto culture dishes coated with 10 µg/ml ECM proteins. Adhesion to PBS, collagen I (Coll I), Fibronectin (FN), or Laminin (Lam) was assayed for 15 minutes (C) or 45 minutes (D). One representative experiment out of three is shown. Results from triplicate wells are displayed as means±SEM (ns, not significant; *, P<0.05; **, P<0.01). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24475194), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human AGER by Immunohistochemistry Differential expression of RAGE and S100A8/A9 on protein level in whole skin.The expression level was analysed by immunochistochemistry using specific anti human S100A8/A9 and anti human RAGE antibodies. The expression was tested in 5 patient samples per group (IC and OTR with in situ or invasive SCC). a: Expression of RAGE and S100A8/A9 in IC and OTR patients with in situ SCC in comparison to normal skin. b: Expression of RAGE and S100A8/A9 in IC and OTR patient with invasive SCC in comparison to normal skin. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0120971), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human AGER by Flow Cytometry Exogenous S100A8/A9 induces keratinocyte proliferation.a: Assessment of the proliferation by BrdU assay. To assess the role of S100A8/A9 in normal primary, AK and SCC cultures, they were treated with purified S100A8/A9 for 24 hours and proliferation was assessed by BrdU incorporation. The induction of cellular proliferation was between 30–70% (2 way Anova, p<0.0001). b: Assessment of the proliferation by the incorporation of BrdU analysed by flow cytrometry. Normal keratinocytes were treated with S100A8/A9 (10ng/ml) and BrdU for 24 hours. Afterwards cells were fixed in 4% PFA, permeabilized with 0.5% Triton and stained for RAGE and BrdU using the antibodies mentioned above. The differential BrdU incorporation was compared to untreated cell. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0120971), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human AGER by Western Blot RAGE expression contributes to cell-cell adhesion.(A) Western blot analysis on control HEK cells (HEK/pcDNA) or expressing FL-RAGE (HEK/FL-RAGE) using anti-RAGE N-term1 antibody. Forty µg of cell lysate were loaded and detection of GAPDH was used as loading control. (B) A dissociation assay was performed by applying mechanical force on monolayers of HEK/pcDNA or HEK/FL-RAGE cells. Photographs were taken at 40× magnification in phase contrast. Bar corresponds to 50 µm. The dissociation index is the means±SEM of three independent experiments (**, P<0.01). (C) Localization of FL-RAGE on HEK/pcDNA, HEK/FL-RAGE or a mix of both cell lines (orange). Nuclei are stained in blue. Red arrows indicate HEK/pcDNA cells in contact with HEK/FL-RAGE. Green arrows indicate HEK/FL-RAGE cells in contact with each other. Bar corresponds to 10 µm. (D) Localization of FL-RAGE on R3/1-pLXSN, R3/1-FL-RAGE or a mix of both cell lines (orange). Nuclei are stained in blue. Red arrows indicate R3/1-pLXSN cells in contact with R3/1-FL-RAGE. Green arrows indicates R3/1-FL-RAGE cells in contact with each other. Red arrows indicate cells not expressing RAGE in contact with cells expressing RAGE. Bar corresponds to 10 µm. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24475194), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human AGER by Western Blot The receptor RAGE critically mediates the cellular response to S100A8/A9.a: Direct blockade by a specific RAGE blocking antibody reduces cellular proliferation. RAGE dependent proliferation was analyzed in normal primary, AK and SCC cells. Cells were incubated for 1 hour with a blocking anti-RAGE antibody (80μg/ml, as recommended by manufacturer) followed by S100A8/A9 stimulation for additional 24 hours. The differences in the proliferation after the blockade were assessed by BrdU incorporation (1 way Anova, Bonferroni`s Multiple test, **p<0.05, ***p<0.05). b: Knockdown of RAGE using shRNA reduces cellular proliferation. The knockdown studies were performed using specific lentiviral shRNA against RAGE. Primary keratinocytes were infected by shRAGE and sh control viral particles. Selection of positive clones was performed by puromycine selection. Cells were grown to optimal confluence and were stimulated with 10ng/ml S100A8/A9.Cells with RAGE knockdown showed a delay in proliferation in comparison to control as assessed by BrdU (70%) (1 way Anova, Bonferroni`s Multiple test p****<0.0001) and microscope images. Exogenous S100A8/A9 did not induce proliferation of shRAGE keratinocytes, but only in the control. c: Blocking TLR4 using specific blocking antibody (HTA125) does not impair cellular proliferation. AK cells were treated with a specific blocking TLR4 antibody (HTA125). AK cells were grown for 24 hours in the presence of HTA125 antibody (1μg/ml) and S100A8/A9 (10ng/ml). For detection of the cellular proliferation rate BrdU proliferation assay was performed. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0120971), licensed under a CC-BY license. Not internally tested by R&D Systems.