pS363-DOP (phospho-∂-Opioid Receptor Antibody)

Figure 1. Agonist-induced Serine363 phosphorylation of the δ-Opioid receptor. Upper panel, HEK293 cells stably expressing the δ-Opioid Receptor (DOP) were either not exposed or exposed to 10 μM DPDPE ([D-Pen2,D-Pen5]-enkephalin) or 0.1 μM PMA (Phorbol 12-Myristate 13-Acetate) for 30 minutes. Cells were lysed and immunoblotted with the anti-pS363-DOP antibody (7TM0317B) at a dilution of 1:1000. Lower panel, blot was stripped and reprobed with a phosphorylation-independent anti-DOP antibody to confirm equal loading of the gel.

Figure 2. Analysis of dose-dependent δ-Opioid Receptor phosphorylation using two phosphosite-specific antibodies. Upper two panels, HEK293 cells stably expressing the δ-Opioid Receptor (DOP) were either not exposed or exposed to increasing concentrations of selective δ-opioid receptor agonist SNC80 ranging from 1 nM to 10 μM for 30 minutes. Cells were lysed and immunoblotted with the anti-pT361-DOP antibody (7TM0317A) or anti-pS363-DOP antibody (7TM0317B) at a dilution of 1:1000. Lower panel, blot was stripped and reprobed with a phosphorylation-independent anti-DOP antibody to confirm equal loading of the gel.

Figure 3. Immunocytochemical identification of Serine363 phosphorylation of the δ-Opioid Receptor. HEK293 cells stably expressing the δ-Opioid Receptor (DOP) were either not exposed or exposed to 10 μM DPDPE ([D-Pen2,D-Pen5]-enkephalin) for 2, 5 or 30 min and immunocytochemically stained with the anti-pS363-DOP antibody (7TM0317B) at a dilution of 1:200. Note, Serine363-phosphorylated DOP receptors were not detectable in untreated cells (0 min). Serine363-phosphorylated DOP receptors were seen at the plasma membrane (2 min) and with increased time of stimulation (5 and 30 min) as perinuclear clusters.

Mann A, Liebetrau S, Klima M, Dasgupta P, Massotte D, Schulz S. Agonist-induced phosphorylation bar code and differential post-activation signaling of the delta opioid receptor revealed by phosphosite-specific antibodies. Sci Rep. 2020 May 22;10(1):8585. doi: 10.1038/s41598-020-65589-7. PMID: 32444688