![]() ![]() However, the antibodies that were validated for flow cytometry are not guaranteed to work for microscopy. At the time this was written, a majority of commercially available antibodies for TSHR have epitopes on the extracellular domain, and several IGF1R antibodies for the α-subunit are available. Notably, PLA experiments in our lab using IGF1R antibodies that bind to the α and β subunits were unsuccessful (unpublished) despite the fact that homodimer formation is an integral characteristic of IGF1R. Even if cytoplasmic and extracellular epitopes are closer than 40 nm apart, PLA probes cannot cross the plasma membrane. Therefore, the antibody epitopes must either be both in the cytoplasmic side or the extracellular side. Unlike experiments with proteins in solution, the spatial orientation of receptors within the plasma membrane remains intact. Therefore, PLA can be used to narrow down which model most likely applies to the disease being studied.Īdapting PLA to study receptor signaling required additional considerations for antibody selection. Various models of receptor crosstalk have been described ( 19, 20), and not all require receptors to be proximal to each other. However, the molecular details of the apparent synergy that mediates this effect is not fully elucidated. ![]() To summarize what has been recently reviewed ( 11, 12), IGF1R inhibitors may partially inhibit TSHR signaling ( 13– 15) simultaneous activation of TSH and IGF1 receptors can lead to greater-than-additive downstream signals ( 16, 17) and some TSHR stimulating antibodies may initiate both IGF1R-independent and -dependent pathways ( 18). The involvement of TSHRs and IGF1Rs acting together to regulate responses in thyrocytes and OFs has been well-documented ( 10). We will then detail the procedures used to detect receptor closeness using in situ Duolink PLA. In the following sections, we will briefly review how PLA was used to extend our understanding of TSHR signaling and describe considerations that were taken into account when adapting PLA to our specific experimental systems. Specifically, we studied TSHR homodimerization and TSHR/IGF1R crosstalk. We were able to study these receptor interactions primarily using endogenously expressed receptors and used overexpression of TSHR to complement these findings. We elected to study potential interactions of TSH receptors (TSHRs) and IGF-1 receptors (IGF1Rs) in primary cultures of human thyroid cells and of human orbital fibroblasts/pre-adipocytes (OFs) as those play a pivotal role in the pathogenesis of Graves’ disease and associated thyroid eye disease. Since overexpression can be associated with aberrant protein localization and interactions ( 9), it is often better to study endogenously expressed proteins. It was used in experiments in SW1736 human anaplastic thyroid and HeLa human cervical cancer cell lines that were made to overexpress NIS ( 8). ![]() To our knowledge, except for our studies, PLA has only been used in the field of thyroid research to show dimerization of the sodium-iodide symporter (NIS). For these reasons, PLA is better suited for studies of endogenously expressed proteins in primary cells. Compared to Co-IP, PLA can be scaled down in terms of number of cells and amount of reagents. Since this method uses fluorescent microscopy, information on subcellular location can also be collected. In contrast, PLA is done after fixation, which could capture transient events and preserve low-affinity interactions. Transient, low-affinity interactions, which are common in GPCR signaling, are difficult to detect with Co-IP. For this method to work, interactions between proteins in the complex must be strong enough so that their affinity is maintained while in solution. These binding partners are later identified with Western blotting. An antibody pulls down one protein member and its binding partners. Co-IP is a technique used to identify protein-protein interactions by using target protein-specific antibodies to capture proteins that are bound to a specific target protein. Both PLA and Co-IP depend on antibodies that recognize proteins in the complex. In situ PLA has some advantages over methods such as co-immunoprecipitation (Co-IP). ![]()
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |