C5a1 Sample Pack (phospho- and non-phospho-C5a1 Receptor Antibodies)

Figure 1. Agonist-induced Threonine324/Serine327 phosphorylation of the Complement C5a Receptor 1. Upper panel, HEK293 cells stably expressing the Complement C5a Receptor 1 (C5a1) were either not exposed or exposed to 1 µM specific Complement C5a receptor agonist CO28 or 10 μM Forskolin or 0.1 μM PMA (Phorbol 12-Myristate 13-Acetate) for 30 minutes. Cells were lysed and immunoblotted with the anti-pT324/pS327-C5a1 antibody (7TM0032A) at a dilution of 1:1000. Lower panel, blot was stripped and reprobed with the phosphorylation-independent anti-C5a1 antibody (7TM0032N-WB) at a dilution of 1:1000 to confirm equal loading of the gel.

Figure 2. Agonist-induced Serine332/Serine334 phosphorylation of the Complement C5a Receptor 1. Upper panel, HEK293 cells stably expressing the Complement C5a Receptor 1 (C5a1) were either not exposed or exposed to 1 µM specific Complement C5a receptor agonist CO28 or 10 μM Forskolin or 0.1 μM PMA (Phorbol 12-Myristate 13-Acetate) for 30 minutes. Cells were lysed and immunoblotted with the anti-pS332/pS334-C5a1 antibody (7TM0032B) at a dilution of 1:1000. Lower panel, blot was stripped and reprobed with the phosphorylation-independent anti-C5a1 antibody (7TM0032N-WB) at a dilution of 1:1000 to confirm equal loading of the gel.

Figure 3. Agonist-induced Threonine336 phosphorylation of the Complement C5a Receptor 1. Upper panel, HEK293 cells stably expressing the Complement C5a Receptor 1 (C5a1) were either not exposed or exposed to 1 µM specific Complement C5a receptor agonist CO28 or 0.1 μM PMA (Phorbol 12-Myristate 13-Acetate) for 30 minutes. Cells were lysed and immunoblotted with the anti-pT336-C5a1 antibody (7TM0032C) at a dilution of 1:1000. Lower panel, blot was stripped and reprobed with the phosphorylation-independent anti-C5a1 antibody (7TM0032N-WB) at a dilution of 1:1000 to confirm equal loading of the gel.

Figure 4. Agonist-induced Serine338/Threonine339 phosphorylation of the Complement C5a Receptor 1. Upper panel, HEK293 cells stably expressing the Complement C5a Receptor 1 (C5a1) were either not exposed or exposed to 1 µM specific Complement C5a receptor agonist CO28 or 0.1 μM PMA (Phorbol 12-Myristate 13-Acetate) for 30 minutes. Cells were lysed and immunoblotted with the anti-pS338pT339-C5a1 antibody (7TM0032D) at a dilution of 1:1000. Lower panel, blot was stripped and reprobed with the phosphorylation-independent anti-C5a1 antibody (7TM0032N-WB) at a dilution of 1:1000 to confirm equal loading of the gel.

Figure 5. Agonist-induced Threonine342 phosphorylation of the Complement C5a Receptor 1. Upper panel, HEK293 cells stably expressing the Complement C5a Receptor 1 (C5a1) were either not exposed or exposed to 1 µM specific Complement C5a receptor agonist CO28 or 0.1 μM PMA (Phorbol 12-Myristate 13-Acetate) for 30 minutes. Cells were lysed and immunoblotted with the anti-pT342-C5a1 antibody (7TM0032E) at a dilution of 1:1000. Lower panel, blot was stripped and reprobed with the phosphorylation-independent anti-C5a1 antibody (7TM0032N-WB) at a dilution of 1:1000 to confirm equal loading of the gel.

Figure 6. Immunohistochemical identification of Complement C5a Receptor 1 in human spleen. Sections were dewaxed, microwaved in citric acid, and incubated with anti-C5a1 (Complement C5a Receptor 1) antibody (7TM0032N-IC) at a dilution of 1:100. Sections were then sequentially treated with biotinylated anti-rabbit IgG and avidin-biotin solution.Color was developed by incubation in 3-amino-9-ethylcarbazole (AEC), and sections were counterstained with hematoxylin. Note, C5a1 receptors were detected at the plasma membrane of a distinct population of cells in human spleen.

Figure 7. Immunohistochemical identification of Complement C5a Receptor 1 in mouse spleen. Sections were dewaxed, microwaved in citric acid, and incubated with anti-C5a1 (Complement C5a Receptor 1) antibody (7TM0032MN-IC) at a dilution of 1:100. Sections were then sequentially treated with biotinylated anti-rabbit IgG and avidin-biotin solution. Color was developed by incubation in 3-amino-9-ethylcarbazole (AEC), and sections were counterstained with hematoxylin. Note, C5a1 receptors were detected at the plasma membrane of nearly all cells in human spleen.

Nürge B, Schulz AL, Kaemmerer D, Sänger J, Evert K, Schulz S, Lupp A. Immunohistochemical identification of complement peptide C5a receptor 1 (C5aR1) in non-neoplastic and neoplastic human tissues. PLoS One. 2021 Feb 19;16(2):e0246939. doi: 10.1371/journal.pone.0246939. PMID: 33606748; PMCID: PMC7894821.

Kaufmann J, Blum NK, Nagel F, Schuler A, Drube J, Degenhart C, Engel J,Eickhoff JE, Dasgupta P, Fritzwanker S, Guastadisegni M, Schulte C, Miess-Tanneberg E, Maric HM, Spetea M, Kliewer A, Baumann M, Klebl B, Reinscheid RK,Hoffmann C, Schulz S. A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening. Commun Biol. 2022 Nov 9;5(1):1206. doi: 10.1038/s42003-022-04135-9. PMID: 36352263; PMCID: PMC9646841.