Visualization of Ceramide-Associated Proteins in Ceramide-Rich Platforms Using a Cross-Linkable Ceramide Analog and Proximity Ligation Assays With Anti-ceramide Antibody.

ABSTRACT: Ceramide-rich platforms (CRPs) mediate association of proteins with the sphingolipid ceramide and may regulate protein interaction in membrane contact sites to the cytoskeleton, organelles, and infectious pathogens. However, visualization of ceramide association to proteins is one of the greatest challenges in understanding the cell biology of ceramide. Here we introduce a novel labeling technique for ceramide-associated proteins (CAPs) by combining photoactivated cross-linking of a bioorthogonal and bifunctional ceramide analog, pacFACer with proximity ligation assays (PLAs). pacFACer cross-linked to CAPs is covalently attached to a fluorophore using click chemistry. PLAs use antibodies to: (1) the candidate CAP and the fluorophore (PLA1); and (2) the CAP and ceramide (PLA2). PLA1 shows t

SUBMITTER: Jiang X 

PROVIDER: S-EPMC6706757 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature