pT362/pS363-NOP (phospho-Nociceptin/Orphanin FQ Receptor Antibody)

Figure 1. Agonist-induced Threonine362/Serine363 phosphorylation of the Nociceptin/Orphanin FQ Receptor. Upper panel, HEK293 cells stably expressing the Nociceptin/Orphanin FQ receptor (NOP) were either not exposed or exposed to 10 μM Nociceptin/Orphanin FQ or 0.1 μM PMA (Phorbol 12-Myristate 13-Acetate) for 30 minutes. Cells were lysed and immunoblotted with the anti-pT362/pS363-NOP antibody (7TM0320C) at a dilution of 1:1000. Lower panel, blot was stripped and reprobed with the phosphorylation-independent anti-NOP antibody (7TM0320N-WB) at a dilution of 1:1000 to confirm equal loading of the gel.

Figure 2. Analysis of dose-dependent Nociceptin/Orphanin FQ Receptor phosphorylation using a panel of phosphosite-specific antibodies. Upper three panels, HEK293 cells stably expressing the Nociceptin/Orphanin FQ Receptor (NOP) were either not exposed or exposed to increasing concentrations of Nociceptin/Orphanin FQ ranging from 1 nM to 10 μM for 30 minutes. Cells were lysed and immunoblotted with the anti-pS346-NOP antibody (7TM0320A) or anti-pS351-NOP antibody (7TM0320B) or anti-pT362/pS363-NOP antibody (7TM0320C) at a dilution of 1:1000. Lower panel, blot was stripped and reprobed with the phosphorylation-independent anti-NOP antibody (7TM0320N-WB) at a dilution of 1:1000 to confirm equal loading of the gel.

Figure 3. Analysis of dose-dependent Nociceptin/Orphanin FQ Receptor phosphorylation in vivo in NOP-eGFP knock-in mice using two phosphosite-specific antibodies. Upper two panels, NOP-eGFP knock-in mice stably expressing the Nociceptin/Orphanin FQ Receptor tagged with enhanced green fluorescent protein (NOP-eGFP) were intraperitoneal injected with 0.9% NaCl or increasing concentrations of specific Nociceptin/Orphanin FQ receptor agonist AT-202 ranging from 0.3 to 30 mg/kg for 30 minutes. Mice were euthanized and brains were removed. The NOP-eGFP receptor was immunoprecipitated with anti-GFP protein agarose beads and immunoblotted with the anti-pS351-NOP antibody (7TM0320B) or anti-pT362/pS363-NOP antibody (7TM0320C) at a dilution of 1:1000. Lower panel, blot was stripped and reprobed with the phosphorylation-independent anti-NOP antibody (7TM0320N-WB) at a dilution of 1:1000 to confirm equal loading of the gel.

Mann A, Moulédous L, Froment C, O'Neill PR, Dasgupta P, Günther T, Brunori G, Kieffer BL, Toll L, Bruchas MR, Zaveri NT, Schulz S. Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists. Sci Signal. 2019 Mar 26;12(574). pii: eaau8072. doi: 10.1126/scisignal.aau8072. PubMed PMID: 30914485; PubMed Central PMCID: PMC6934085.

Dasgupta P, Mann A, Polgar WE, Reinscheid RK, Zaveri NT, Schulz S. Attenuated G protein signaling and minimal receptor phosphorylation as a biochemical signature of low side-effect opioid analgesics. Sci Rep. 2022 May 3;12(1):7154. doi: 10.1038/s41598-022-11189-6. PMID: 35504962; PMCID: PMC9065038.