title: Detection of signaling effector-complexes downstream of bmp4 using PLA, a proximity ligation assay.
abstract: BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects (1). Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription. In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity (2-4). This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.
authors.affiliation: Medical Research Council, Clinical Sciences Centre, Imperial College, Hammersmith Hospital.
authors.forename: Efstathia
authors.initials: E
authors.lastname: Thymiakou
authors.forename: Vasso
authors.initials: V
authors.lastname: Episkopou
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D055415:Bone Morphogenetic Protein 4 / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D019556:COS Cells; D002522:Chlorocebus aethiops; D057809:HEK293 Cells; D006801:Humans; D010766:Phosphorylation; D025941:Protein Interaction Mapping / Q000379:methods*; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D051785:Smad Proteins / Q000378:metabolism
chemical_list: D055415:Bone Morphogenetic Protein 4; D011720:Pyrazoles; D011743:Pyrimidines; D051785:Smad Proteins; C516138:dorsomorphin