Human CD47 Antibody

Cell Adhesion Mediated by SIRP alpha/CD172a and Neutral-ization by Human CD47 Antibody.

Recombinant Human SIRPa/CD172a (Catalog # 4546-SA), immobilized onto a microplate, supports the adhesion of the human erythrocytes in a dose-dependent manner (orange line). Adhesion elicited by Recombinant Human SIRP-a (2 µg/mL) is neutralized (green line) by increasing concentrations of Sheep Anti-Human CD47 Poly-clonal Antibody (Catalog # AF4670). The adhesion was maximally inhibited (70-100%) by 2 µg/mL of the antibody.

Western Blot Shows Human CD47 Specificity by Using Knockout Cell Line.

Western blot shows lysates of HEK293T human embryonic kidney parental cell line and CD47 knockout HEK293T cell line (KO). PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human CD47 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4670) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). Specific bands were detected for CD47 at approximately 50 kDa (as indicated) in the parental HEK293T cell line, but is not detectable in knockout HEK293T cell line. GAPDH (Catalog # AF5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human CD47 by Western Blot

Increased CD47 expression in aging and replicative senescence. (A) Quantification of relative CD47 mRNA levels by qPCR. Expression levels of senescent cells were normalized to the respective proliferating control (white bar). Data are representative of three independent experiments. All values are means ± SEM. **P < 0.005, ***P < 0.0005, ****P < 0.0001. Statistically significant differences were determined by unpaired Student’s t test. (B) Representative immunofluorescence images from indicated cell types stained for CD47 (green) and Hoechst (blue). Scale bar: 20 µm, except for 3T3 cells: 50 µm. (C) Whole-cell lysates from indicated cell types were isolated and analyzed by Western blotting for the indicated proteins. Senescence was induced by chemical treatment (palbociclib, except for A549: etopsoside). Shown are representative blots of three independent experiments. GAPDH images are derived from the same blot as Fig. S3 (for Panc1 cells the same GAPDH loading control is additionally used in Fig. 8 G). (D) Representative immunofluorescence images of proliferating or senescent small epithelial cells (SAEC) stained for CD47 (green) and Hoechst (blue). scale bars indicate 20 µm. n = 2–3. (E) Western blot analysis of CD47 expression in SAEC cells. Senescence was induced by chemical treatment (palbociclib). GAPDH was used as loading control. n = 2. GAPDH images are derived from the same blot as Fig. S3. (F) Representative immunofluorescence images of primary lung fibroblast (proliferative or replicative senescent by serially passaging; normal healthy [NHLF] or IPF-derived [IPF]) showing CD47 (green) and Hoechst (blue) staining. scale bars indicate 20 µm. n = 2. (G) Whole-cell lysates from primary NHLF or IPF lung fibroblasts were isolated and analyzed by Western blotting for the indicated proteins. Senescence was induced by replicative passing. Shown are representative blots of three to four independent experiments. Source data are available for this figure: SourceData F7. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36459066), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CD47 by Immunocytochemistry/Immunofluorescence

Adhesion of EpCAMpos/neg cells on multi-marker arrays.2x105 EpCAMpos (cell pool of MCF7, SKBR3, HCC1500, ZR-75-1, TMX2-28) (A) and EpCAMlow/neg cells (MDA-MB-231) (B) were either stained with 1 μM MitoTracker Green FM (A) or MitoTracker Orange CM (B) and incubated for cell adhesion experiments on NEXTERION slides AL, coated with different antibodies and ECM molecules, alone and in combination (0.1 mg/ml each). The labeling above the single spots indicates respective capture molecules (Iso = isotype control, ms = mouse, Lam = laminin, Col = collagen, HA = hyaluronic acid, rt = rat, T = Trop2, EpC = EpCAM, 49f = CD49f, rb = rabbit). (C) Overlay image of (A) and (B); scale bars (white) = 500 μm, 20x magnification. (D) Array layout (5x5 mm) with 36 spots (spot diameter = 500 μm; pitch = 800 μm) printed on NEXTERION slides AL. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26695635), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CD47 by Western Blot

Increased CD47 expression in aging and replicative senescence. (A) Quantification of relative CD47 mRNA levels by qPCR. Expression levels of senescent cells were normalized to the respective proliferating control (white bar). Data are representative of three independent experiments. All values are means ± SEM. **P < 0.005, ***P < 0.0005, ****P < 0.0001. Statistically significant differences were determined by unpaired Student’s t test. (B) Representative immunofluorescence images from indicated cell types stained for CD47 (green) and Hoechst (blue). Scale bar: 20 µm, except for 3T3 cells: 50 µm. (C) Whole-cell lysates from indicated cell types were isolated and analyzed by Western blotting for the indicated proteins. Senescence was induced by chemical treatment (palbociclib, except for A549: etopsoside). Shown are representative blots of three independent experiments. GAPDH images are derived from the same blot as Fig. S3 (for Panc1 cells the same GAPDH loading control is additionally used in Fig. 8 G). (D) Representative immunofluorescence images of proliferating or senescent small epithelial cells (SAEC) stained for CD47 (green) and Hoechst (blue). scale bars indicate 20 µm. n = 2–3. (E) Western blot analysis of CD47 expression in SAEC cells. Senescence was induced by chemical treatment (palbociclib). GAPDH was used as loading control. n = 2. GAPDH images are derived from the same blot as Fig. S3. (F) Representative immunofluorescence images of primary lung fibroblast (proliferative or replicative senescent by serially passaging; normal healthy [NHLF] or IPF-derived [IPF]) showing CD47 (green) and Hoechst (blue) staining. scale bars indicate 20 µm. n = 2. (G) Whole-cell lysates from primary NHLF or IPF lung fibroblasts were isolated and analyzed by Western blotting for the indicated proteins. Senescence was induced by replicative passing. Shown are representative blots of three to four independent experiments. Source data are available for this figure: SourceData F7. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36459066), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CD47 by Immunocytochemistry/Immunofluorescence

Adhesion of EpCAMpos/neg cells on multi-marker arrays.2x105 EpCAMpos (cell pool of MCF7, SKBR3, HCC1500, ZR-75-1, TMX2-28) (A) and EpCAMlow/neg cells (MDA-MB-231) (B) were either stained with 1 μM MitoTracker Green FM (A) or MitoTracker Orange CM (B) and incubated for cell adhesion experiments on NEXTERION slides AL, coated with different antibodies and ECM molecules, alone and in combination (0.1 mg/ml each). The labeling above the single spots indicates respective capture molecules (Iso = isotype control, ms = mouse, Lam = laminin, Col = collagen, HA = hyaluronic acid, rt = rat, T = Trop2, EpC = EpCAM, 49f = CD49f, rb = rabbit). (C) Overlay image of (A) and (B); scale bars (white) = 500 μm, 20x magnification. (D) Array layout (5x5 mm) with 36 spots (spot diameter = 500 μm; pitch = 800 μm) printed on NEXTERION slides AL. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26695635), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CD47 by Immunocytochemistry/Immunofluorescence

Adhesion of EpCAMpos/neg cells on multi-marker arrays.2x105 EpCAMpos (cell pool of MCF7, SKBR3, HCC1500, ZR-75-1, TMX2-28) (A) and EpCAMlow/neg cells (MDA-MB-231) (B) were either stained with 1 μM MitoTracker Green FM (A) or MitoTracker Orange CM (B) and incubated for cell adhesion experiments on NEXTERION slides AL, coated with different antibodies and ECM molecules, alone and in combination (0.1 mg/ml each). The labeling above the single spots indicates respective capture molecules (Iso = isotype control, ms = mouse, Lam = laminin, Col = collagen, HA = hyaluronic acid, rt = rat, T = Trop2, EpC = EpCAM, 49f = CD49f, rb = rabbit). (C) Overlay image of (A) and (B); scale bars (white) = 500 μm, 20x magnification. (D) Array layout (5x5 mm) with 36 spots (spot diameter = 500 μm; pitch = 800 μm) printed on NEXTERION slides AL. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26695635), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CD47 by Western Blot

Impairment of efferocytosis by senescent cells is mediated by CD47 and CD24 expression. (G) Whole-cell lysates derived from Panc1 cells (WT and CD47 KO) were analyzed by Western blotting for the indicated proteins. GAPDH was used as loading control. Senescence was induced by chemical treatment (Palbociclib). Blots are representative of three independent experiments. GAPDH image is derived from the same blot as Fig. 7 and Fig. S3. CD24 image is derived from the same blot as in Fig. S3. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36459066), licensed under a CC-BY license. Not internally tested by R&D Systems.