Anti-FLAG tag antibody, clone O4 [SureLight® P1]

Background: It has been demonstrated that kappa-opioid receptor agonists can reduce hypoxia-ischemia brain injury in animal models. However, it is unclear how the kappa-opioid receptor responds to hypoxia-ischemia. In the current study, the authors used an in vitro model of oxygen-glucose deprivation and reoxygenation to explore how kappa-opioid receptors respond to hypoxia and reoxygenation. Methods: Mouse neuroblastoma Neuro2A cells were stably transfected with mouse kappa-opioid receptor-tdTomato fusion protein or Flag-tagged mouse kappa-opioid receptor, divided into several groups (n = 6 to 12), and used to investigate the kappa-opioid receptor movement. Observations were performed under normal oxygen, at 30 min to 1 h after oxygen-glucose deprivation and at 1 h after reoxygenation using high-resolution imaging techniques including immunoelectronmicroscopy in the presence and absence of kappa-opioid receptor antagonist, dynamin inhibitors, potassium channel blockers, and dopamine receptor inhibitor. Results: Hypoxic conditions caused the kappa-opioid receptor to be internalized into the cells. Inhibition of dynamin by Dyngo-4a prevented the receptor internalization. Interestingly, a specific kappa-opioid receptor antagonist norbinaltorphimine blocked internalization, suggesting the involvement of activation of a specific kappa-opioid receptor. kappa-Opioid receptor internalization appears to be reversed by reoxygenation. Quantities of intracellular kappa-opioid receptor-associated gold particles as demonstrated by immunoelectron microscopy were increased from 37 to 85% (P < 0.01) after oxygen-glucose deprivation. Potassium channel blockers and dopamine receptor inhibitor failed to block hypoxia-induced kappa-opioid receptor internalization. Conclusions: Hypoxia induces reversible kappa-opioid receptor internalization, which was inhibited by selective kappa-opioid receptor antagonists or dynamin inhibitor, and can be reversed by reoxygenation in neuroblastoma cells, indicating the modulating effects between kappa-opioid receptor and hypoxia via kappa-opioid receptor activation and the dynamin-dependent mechanism.

The beta(2)-adrenergic receptor (beta(2)AR) is a prototypical member of the G protein-coupled receptor (GPCR) superfamily of proteins and is one of the best-characterized GPCRs due to its role in several important physiological systems. Because of limited availability of high quality antibodies against GPCRs, much of the work done on beta(2)AR took advantage of heterologous expression systems. Overexpressed proteins may overwhelm the cellular regulatory machinery leading potentially to responses distinct from the native protein. To address this issue we generated a stable cell line with a tetracycline-inducible beta(2)AR tagged with a FLAG epitope, such that we are able to control the quantity of receptor produced. This allows us to induce a discrete pulse of FLAG-beta(2)AR transcription and translation allowing us to follow the complete life cycle of the protein from synthesis as an immature protein to degradation. We show that such limited pulses of receptor expression lead to signaling phenotypes that more closely reflect endogenous signaling events.