In JoVE (1)
Other Publications (1)
Articles by Mayu Otani in JoVE
Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation Jun Imai1, Mayu Otani1, Takahiro Sakai1, Shinichi Hatta1 1Laboratory of Physiological Chemistry, Faculty of Pharmacy, Takasaki University of Health and Welfare The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.
Other articles by Mayu Otani on PubMed
Purification of the Subcellular Compartment in Which Exogenous Antigens Undergo Endoplasmic Reticulum-associated Degradation from Dendritic Cells Heliyon. Sep, 2016 | Pubmed ID: 27656684 Dendritic cells (DCs) are capable of processing and presenting exogenous antigens using MHC class I molecules. This pathway is called antigen cross-presentation and plays an important role in the stimulation of naïve CD8(+) T cells for infectious and tumor immunity. Our previous studies in DC2.4 cells and bone marrow-derived DCs revealed that exogenously added ovalbumin (OVA) is processed through endoplasmic reticulum (ER)-associated degradation (ERAD) for cross-presentation. In this study, we aimed to further confirm these results by purification of the subcellular compartment in which exogenous antigens undergo ERAD from homogenates of DC2.4 cells pretreated with biotinylated OVA (bOVA). bOVA-containing vesicles were purified using streptavidin (SA)-magnetic beads from cell homogenates and were found to contain ER chaperones and ERAD components together with proteins for antigen presentation. In purified microsomes, bOVA was retained in membranous fractions and degraded by the ubiquitin proteasome system in presence reticulocyte lysates and ATP. These results strongly suggested that DCs processed and degraded exogenous antigens through ERAD for cross-presentation in this purified subcellular compartment.