Assessment of Protein Expression by Proximity Ligation Assay in the Nonhuman Primate Endometrium, Placenta, and Fetal Adrenal in Response to Estrogen

Methods Mol Biol. 2016;1366:149-161. doi: 10.1007/978-1-4939-3127-9_12.

Abstract

In the field of protein biology, immunology-based techniques have been evolving for detection and quantification of protein levels, protein-protein interaction, and protein modifications in cells and tissues. The proximity ligation assay (PLA), a method of detection that combines immunologic and PCR-based approaches, was developed to overcome some of the drawbacks that are inherent to other detection methods. The PLA allows for very sensitive and discretely quantifiable measures of unmodified, native protein levels, and protein-protein interaction/modification complexes in situ in both fixed tissues and cultured cells. We describe herein the PLA method and its applicability to quantify the effects of estrogen on expression of angioregulatory factors, e.g., angiopoietin-1 (Ang-1) in the endometrium, vascular endothelial growth factor (VEGF) in the placenta, and melanocortin 2 receptor (MC2R)/accessory protein (MRAP) in the fetal adrenal of the nonhuman primate.

Keywords: Adrenal; Endometrium; Estrogen; Immunocytochemistry; Immunofluorescence; Placenta; Proximity ligation assay.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adrenal Glands / drug effects*
  • Adrenal Glands / embryology
  • Adrenal Glands / immunology
  • Adrenal Glands / metabolism
  • Angiopoietin-1 / metabolism
  • Animals
  • Antibodies / immunology
  • Antibodies / metabolism
  • Antibody Specificity
  • Endometrium / drug effects*
  • Endometrium / immunology
  • Endometrium / metabolism
  • Estrogens / pharmacology*
  • Female
  • Fluorescent Antibody Technique
  • Microscopy, Fluorescence
  • Oligonucleotides / metabolism
  • Papio
  • Placenta / drug effects*
  • Placenta / immunology
  • Placenta / metabolism
  • Polymerase Chain Reaction
  • Pregnancy
  • Protein Binding
  • Protein Interaction Mapping*
  • Proteins / immunology
  • Proteins / metabolism*
  • Proteomics / methods*
  • Receptor, Melanocortin, Type 2 / metabolism
  • Vascular Endothelial Growth Factor A / metabolism
  • Workflow

Substances

  • Angiopoietin-1
  • Antibodies
  • Estrogens
  • Oligonucleotides
  • Proteins
  • Receptor, Melanocortin, Type 2
  • Vascular Endothelial Growth Factor A