A Nanobody-Based Proximity Ligation Assay Detects Constitutive and Stimulus-Regulated Native Arc/Arg3.1 Oligomers in Hippocampal Neuronal Dendrites.

ABSTRACT: Activity-regulated cytoskeleton-associated protein (Arc), the product of an immediate early gene, plays critical roles in synaptic plasticity and memory. Evidence suggests that Arc function is determined by its oligomeric state; however, methods for localization of native Arc oligomers are lacking. Here, we developed a nanobody-based proximity ligation assay (PLA) for detection, localization, and quantification of Arc-Arc complexes in primary rat hippocampal neuronal cultures. We used nanobodies with single, structurally defined epitopes in the bilobar Arc capsid domain. Nanobody H11 binds inside the N-lobe ligand pocket, while nanobody C11 binds to the C-lobe surface. For each nanobody, ALFA- and FLAG-epitope tags created a platform for antibody binding and PLA. Surprisingly, PLA puncta i

SUBMITTER: Baldinotti R 

PROVIDER: S-EPMC11880080 | biostudies-literature | 2025 Apr

REPOSITORIES: biostudies-literature