PMID- 28413811 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20220210 IS - 2331-8325 (Print) IS - 2331-8325 (Electronic) IS - 2331-8325 (Linking) VI - 6 IP - 20 DP - 2016 Oct 20 TI - Cell Surface Protein Detection to Assess Receptor Internalization. LID - e1968 [pii] LID - 10.21769/BioProtoc.1968 [doi] AB - The migration of membrane receptors upon exposure to different stimulants/inhibitors is of great importance. Among others, the internalization of membrane receptors affects their accessibility to ligands and cell responsiveness to environmental cues. Experimentally, receptor internalization can be used as a measure of their activation. In our studies, we employed this approach to explore cross-talk between a seven transmembrane domain receptor for neuropeptide Y (NPY), Y5R, and a tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF), TrkB. To this end, we measured the internalization of Y5R upon stimulation with the TrkB ligand, BDNF. Upon treatment with BDNF, the cells were exposed to a membrane impermeable, biotinylation reagent that selectively labels surface proteins. Subsequently, the biotinylated membrane proteins were affinity-purified on columns with avidin resins and analyzed by Western blot. Differences in the fraction of receptors present on the cell surface of control and ligand-treated cells served as a measure of their internalization and response to particular stimuli. FAU - Czarnecka, Magdalena AU - Czarnecka M AD - Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington DC, USA. FAU - Kitlinska, Joanna AU - Kitlinska J AD - Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington DC, USA. LA - eng GR - R01 CA123211/CA/NCI NIH HHS/United States GR - R01 CA197964/CA/NCI NIH HHS/United States GR - R03 CA178809/CA/NCI NIH HHS/United States GR - R21 CA198698/CA/NCI NIH HHS/United States PT - Journal Article PL - United States TA - Bio Protoc JT - Bio-protocol JID - 101635102 PMC - PMC5390524 MID - NIHMS849927 EDAT- 2017/04/18 06:00 MHDA- 2017/04/18 06:01 PMCR- 2017/04/13 CRDT- 2017/04/18 06:00 PHST- 2017/04/18 06:00 [entrez] PHST- 2017/04/18 06:00 [pubmed] PHST- 2017/04/18 06:01 [medline] PHST- 2017/04/13 00:00 [pmc-release] AID - e1968 [pii] AID - 10.21769/BioProtoc.1968 [doi] PST - ppublish SO - Bio Protoc. 2016 Oct 20;6(20):e1968. doi: 10.21769/BioProtoc.1968.