Detection of signaling effector-complexes downstream of bmp4 using PLA, a proximity ligation assay

J Vis Exp. 2011 Mar 3;(49):2631. doi: 10.3791/2631.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Animals
  • Biological Assay / methods*
  • Bone Morphogenetic Protein 4 / antagonists & inhibitors
  • Bone Morphogenetic Protein 4 / metabolism*
  • Bone Morphogenetic Protein 4 / pharmacology
  • COS Cells
  • Chlorocebus aethiops
  • HEK293 Cells
  • Humans
  • Phosphorylation
  • Protein Interaction Mapping / methods*
  • Pyrazoles / pharmacology
  • Pyrimidines / pharmacology
  • Smad Proteins / metabolism

Substances

  • Bone Morphogenetic Protein 4
  • Pyrazoles
  • Pyrimidines
  • Smad Proteins
  • dorsomorphin