Human CMG-2/ANTXR2 Antibody

Detection of CMG‑2/ANTXR2 in Human Monocytes by Flow Cytometry. Human whole blood monocytes were stained with Human CMG-2/ANTXR2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2940, filled histogram) or control antibody (Catalog # AB-108-C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107).

Detection of Human CMG-2/ANTXR2 by Western Blot Number and localization of glycan sidechains determine trafficking efficiency of TEM8 and CMG2.A) Endoglycosidase H (EndoH) treatment on TEM8 and CMG2 single mutants. HeLa cells were transfected for 48h with the respective cDNAs. 40 μg of cell extracts were treated or not with EndoH as described before. Samples were analyzed by SDS-PAGE and Western Blotting. B) Quantification of surface biotinylation experiments to determine amount of TEM8 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n ≥ 3. * p≤0.05, ** p≤0.01, *** p≤0.001 C) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against TEM8-HA. D) Quantification of surface biotinylation experiments to determine amount of CMG2 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n ≥ 3. * p≤0.05, ** p≤0.01, *** p≤0.001 E) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against CMG2-V5. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25781883), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CMG-2/ANTXR2 by Western Blot Number and localization of glycan sidechains determine trafficking efficiency of TEM8 and CMG2.A) Endoglycosidase H (EndoH) treatment on TEM8 and CMG2 single mutants. HeLa cells were transfected for 48h with the respective cDNAs. 40 μg of cell extracts were treated or not with EndoH as described before. Samples were analyzed by SDS-PAGE and Western Blotting. B) Quantification of surface biotinylation experiments to determine amount of TEM8 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n ≥ 3. * p≤0.05, ** p≤0.01, *** p≤0.001 C) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against TEM8-HA. D) Quantification of surface biotinylation experiments to determine amount of CMG2 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n ≥ 3. * p≤0.05, ** p≤0.01, *** p≤0.001 E) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against CMG2-V5. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25781883), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CMG-2/ANTXR2 by Immunoprecipitation Glycosylation acts as a buffer for CMG2 ectodomain mutations.A) Graphic showing the disulfide bridge C39-C218 (blue) present in CMG2 WT B) Fibroblast cells were treated or not with tunicamycin for 16h and TCE were analyzed by SDS-PAGE and Western Blot. Representative Western Blot with control fibroblasts and patient fibroblasts. Calnexin serves as a loading control. C) Quantification of total protein levels. CMG2 levels are normalized to WT protein level without tunicamycin treatment, which was set to 100%. Statistics were calculated using an unpaired t-test. Errors represent standard deviation. n ≥ 3. * p≤0.05 Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25781883), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CMG-2/ANTXR2 by Immunoprecipitation Loss of glycosylation affects binding of Anthrax toxin to TEM8 but not to CMG2.A and C) HeLa cells were transfected for 48h with the respective cDNAs. Cells were treated for 1h at 4°C with 500 ng/ml PA83 and shifted to 37°C for 10 min to induce cleavage and heptamerization. Immunoprecipitates against TEM8-HA/CMG2-V5 were analyzed by SDS-PAGE and Western Blotting against PA and TEM8-HA/CMG2-V5. Control cells are non-transfected. D50A is a binding deficient mutant that serves as a negative control. B and D) HeLa cells were transfected for 48h with the respective cDNAs. Cells were lysed and incubated for 1h at 4°C with 1 μg/ml PA83. Immunoprecipitates against TEM8-HA/CMG2-V5 were analyzed by SDS-PAGE and Western Blotting against PA and TEM8-HA/CMG2-V5. Control cells are non-transfected. D50A is a binding deficient mutant that serves as a negative control. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25781883), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CMG-2/ANTXR2 by Immunoprecipitation CMG2 and TEM8 can undergo N-glycosylation on all predicted sites.A) Graphics depicting glycosylation sites on TEM8 and CMG2. Sites in red are unique to the respective proteins, N260 in CMG2 (yellow) corresponds to N262 in TEM8. B) Expression of TEM8 glycosylation mutants in HeLa cells. Cells were transfected for 48h with the respective cDNAs. Expression was analyzed by SDS-PAGE and Western Blotting. C) Expression of all CMG2 glycosylation mutants in HeLa cells. Cells were transfected for 48h with the respective cDNAs. Expression was analyzed by SDS-PAGE and Western Blotting. D) Endoglycosidase F (NGaseF) treatment on TEM8 glycosylation mutants. 40 μg of cell extracts were treated or not with NGaseF and analyzed by SDS-PAGE and Western Blotting. E) Endoglycosidase F (NGaseF) treatment on CMG2 glycosylation mutants. 40 μg of cell extracts were treated or not with NGaseF and analyzed by SDS-PAGE and Western Blotting. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25781883), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CMG-2/ANTXR2 by Western Blot Non-glycosylated TEM8 is an ER quality control and ERAD substrate.A) HeLa cells were transfected for 48h with the respective cDNAs. Cells were treated or not with MG132, an inhibitor of the proteasome or Bafilomycin A1, a drug preventing endosomal acidification and thus lysosomal degradation. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and Western Blotting against Ubiquitin and TEM8-HA. B) HEK cells stably expressing CMG2 under the control of a tetracycline inducible promotor were induced for 24h with 0.1μg/ml doxycycline. Cells were treated or not with MG132 or Bafilomycin A1. Immunoprecipitates against CMG2-V5 were analyzed by SDS-PAGE and Western Blotting against Ubiquitin and CMG2-V5. C) HeLa cells were treated or not with tunicamycin, an antibiotic blocking the co-translational transfer of glycan sidechains in the ER by blocking the oligosaccharyltransferase (OST) for 16h. Surface proteins were labeled with biotin and immunoprecipitates against streptavidin were analysed for TEM8 or Calnexin as a negative control. D) RpeI cells were treated or not with tunicamycin for 16h. Surface proteins were labeled with biotin and immunoprecipitates against streptavidin were analysed for CMG2 or Calnexin as a negative control. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25781883), licensed under a CC-BY license. Not internally tested by R&D Systems.