Fig. 5: Differential involvement of GRK2/3 and GRK5/6 in C5a1 multisite phosphorylation. | Communications Biology

Fig. 5: Differential involvement of GRK2/3 and GRK5/6 in C5a1 multisite phosphorylation.

From: A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening

Fig. 5

a Schematic representation showing C5a1-binding sites for mouse anti-hemagglutinin (HA) magnetic beads, rabbit anti-HA antibodies and antibodies against phosphorylated pT324/pS327-, pS332/pS334-, pS338/pT339-, and C5a1 independent of phosphorylation status (np-C5a1). S/T sites detected using phosphosites-specific C5a1 antibodies are depicted in red. All other potential phosphate acceptor sites are depicted in gray. b–d Control-HEK293 cells (gray), ΔGRK2/3-HEK293 cells (orange) and ΔGRK5/6-HEK293 cells (indigo) stably expressing C5a1 were exposed to increasing concentrations of the synthetic agonist C028 and incubated for 30 min at 37 °C. Phosphorylation of T324/S327 (b), S332/S334 (c) and S338/T339 (d) was assessed according to the standard protocol. Means of n = 5 independent experiments performed in duplicates ± SEM are presented in concentration–response curves. Where error bars are not apparent, SEM was smaller than symbol size. All data points were normalized to 10 µM C028 stimulation of C5a1-expressing Control-HEK293 cells.

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