pT324/pS327-C5a1 (phospho-Complement C5a Receptor 1 Antibody)

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. Analysis of dose-dependent Complement C5a Receptor 1 phosphorylation using two phosphosite-specific antibodies. Upper two panels, HEK293 cells stably expressing the Complement C5a Receptor 1 (C5a1) were either not exposed or exposed to increasing concentrations of complement fragment C5a ranging from 1 nM to 100 nM for 30 minutes. Cells were lysed and immunoblotted with the anti-pT324/pS327-C5a1 antibody (7TM0032A) or 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. Analysis of agonist-dependent Complement C5a Receptor 1 phosphorylation using two phosphosite-specific antibodies. Upper two panels, HEK293 cells stably expressing the Complement C5a receptor 1 (C5a1) were either not exposed or exposed to 10 µM CO28 or 100 nM complement fragment C5a or 100 nM des-Arg C5a (C5a lacking the c-terminal arginine) for 30 minutes. Cells were lysed and immunoblotted with the anti-pT324/pS327-C5a1 antibody (7TM0032A) or 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 4. Immunocytochemical identification of Threonine324/Serine327 phosphorylation of the Complement C5a Receptor 1 in HEK293 cells. HEK293 cells stably expressing the Complement C5a Receptor 1 (C5a1) were either not exposed or exposed to 100 nM complement C5a and immunocytochemically stained with the anti-pT324/pS327-C5a1 antibody (7TM0032A) at a dilution of 1:200. Note, Threonine324/Serine327-phosphorylated C5a1 receptors were not detectable in untreated cells (0 min). Threonine324/Serine327-phosphorylated C5a1 receptors were seen as perinuclear clusters of vesicles after 30 min.

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