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IHC-Validated Phosphosite-Specific 7TM Antibodies

G protein-coupled receptors (GPCRs) are vital signal transducers that upon activation become phosphorylated on intracellular serine and threonine residues. However, GPCR phosphorylation is very unstable during routine immunohistochemical procedures, necessitating the presence of appropriate phosphatase inhibitors throughout both fixation and staining procedures. We have therefore developed a protocol that facilitates the visualization of GPCR activation in response to physiological and pharmacological stimulation.

Reference:

Fritzwanker S, Nagel F, Kliewer A, Schulz S. In situ Visualization of therapeutic drug effects using phosphosite-specific GPCR antibodies. bioRxiv 2022.06.14.496067; doi: https://doi.org/10.1101/2022.06.14.496067

Figure 1. In situ visualisation of µ-opioid receptor activation.

Figure 2. In situ visualisation of CB1 cannabinoid receptor activation.

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Immunohistochemical Identification of Serine594/Threonine597/Serine599 phosphorylation of the SMO Receptor in primary cilia in the mouse neural tube
pS594/pT597/pS599-SMO (phospho-SMO Receptor...
Serine594/Threonine597/Serine599 (S362/S363/S364) is major GRK2 phosphorylation site of the SMO receptor. The pS594/pT597/pS599-SMO antibody detects phosphorylation in response to agonists. The pS594/pT597/pS599-SMO antibody can be used...
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Immunohistochemical Identification of Agonist-induced Serine425 phosphorylation of the Cannabinoid Receptor 1 in mouse brain
pS425-CB1 (IHC-grade phospho-Cannabinoid...
Serine425 (S425) is major phosphorylation site of the Cannabinoid Receptor 1 (CB1). The pS425-CB1 antibody detects phosphorylation in response to agonists. S425 phosphorylation is likely to be involved in efficient ligand sequestration...
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KO-Validated
Immunohistochemical Identification of Agonist-induced Serine375 phosphorylation of the µ-Opioid Receptor in mouse spinal cord
pS375-MOP (IHC-grade phospho-µ-Opioid Receptor...
Serine375 (S375) is the primary phosphorylation site in a hierarchical phosphorylation cascade. The pS375-MOP antibody detects phosphorylation in response to high- and low-efficacy agonists but not after PKC activation. S375...
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KO-Validated
Immunohistochemical Identification of Agonist-induced Threonine376 phosphorylation of the µ-Opioid Receptor in mouse spinal cord
pT376-MOP (IHC-grade phospho-µ-Opioid Receptor...
Threonine376 (T376) is a major phosphorylation site of the µ-opioid receptor (MOP). The pT376-MOP antibody detects phosphorylation in response to high-efficacy agonists but to low-efficacy agonists or after PKC activation. T376...
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KO-Validated
Immunohistochemical Identification of Agonist-induced Threonine379 phosphorylation of the µ-Opioid Receptor in mouse spinal cord
pT379-MOP (IHC-grade phospho-µ-Opioid Receptor...
Threonine379 (T379) is a major phosphorylation site of the µ-opioid receptor (MOP). The pT379-MOP antibody detects phosphorylation in response to high-efficacy agonists but to low-efficacy agonists or after PKC activation. T379...
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For further reading refer to:

Fritzwanker S, Nagel F, Kliewer A, Schulz S. In situ Visualization of opioid and cannabinoid drug effects using phosphosite-specific GPCR antibodies. bioRxiv 2022.06.14.496067; doi: https://doi.org/10.1101/2022.06.14.496067

Walker MF, Zhang J, Steiner W, Ku PI, Zhu JF, Michaelson Z, Yen YC, Long AB, Casey MJ, Poddar A, Nelson IB, Arveseth CD, Nagel F, Clough R, LaPotin S, Kwan KM, Schulz S, Stewart RA, Tesmer JJG, Caspary T, Subramanian R, Ge X, Myers BR. A Ciliary SMOOTHENED-GRK2-PKA Signaling Pathway Initiates Hedgehog Signal Transduction. bioRxiv [Preprint]. 2023 May 11:2023.05.10.540226. doi:
10.1101/2023.05.10.540226. PMID: 37214942; PMCID: PMC10197709.

Kliewer A, Reinscheid RK, Schulz S. Emerging Paradigms of G Protein-Coupled Receptor Dephosphorylation. Trends Pharmacol Sci. 2017 Jul;38(7):621-636. doi:10.1016/j.tips.2017.04.002. Epub 2017 May 4. Review. PubMed PMID: 28478994.

For further reading refer to: Fritzwanker S, Nagel F, Kliewer A, Schulz S. In situ Visualization of opioid and cannabinoid drug effects using phosphosite-specific GPCR antibodies. bioRxiv... read more »
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IHC-Validated Phosphosite-Specific 7TM Antibodies

For further reading refer to:

Fritzwanker S, Nagel F, Kliewer A, Schulz S. In situ Visualization of opioid and cannabinoid drug effects using phosphosite-specific GPCR antibodies. bioRxiv 2022.06.14.496067; doi: https://doi.org/10.1101/2022.06.14.496067

Walker MF, Zhang J, Steiner W, Ku PI, Zhu JF, Michaelson Z, Yen YC, Long AB, Casey MJ, Poddar A, Nelson IB, Arveseth CD, Nagel F, Clough R, LaPotin S, Kwan KM, Schulz S, Stewart RA, Tesmer JJG, Caspary T, Subramanian R, Ge X, Myers BR. A Ciliary SMOOTHENED-GRK2-PKA Signaling Pathway Initiates Hedgehog Signal Transduction. bioRxiv [Preprint]. 2023 May 11:2023.05.10.540226. doi:
10.1101/2023.05.10.540226. PMID: 37214942; PMCID: PMC10197709.

Kliewer A, Reinscheid RK, Schulz S. Emerging Paradigms of G Protein-Coupled Receptor Dephosphorylation. Trends Pharmacol Sci. 2017 Jul;38(7):621-636. doi:10.1016/j.tips.2017.04.002. Epub 2017 May 4. Review. PubMed PMID: 28478994.

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