PMID- 28872140
OWN - NLM
STAT- MEDLINE
DCOM- 20180514
LR  - 20190821
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 126
DP  - 2017 Aug 21
TI  - Purification of the Membrane Compartment for Endoplasmic Reticulum-associated 
      Degradation of Exogenous Antigens in Cross-presentation.
LID - 10.3791/55949 [doi]
LID - 55949
AB  - Dendritic cells (DCs) are highly capable of processing and presenting 
      internalized exogenous antigens upon major histocompatibility class (MHC) I 
      molecules also known as cross-presentation (CP). CP plays an important role not 
      only in the stimulation of naive CD8(+) T cells and memory CD8(+) T cells for 
      infectious and tumor immunity but also in the inactivation of self-acting naive T 
      cells by T cell anergy or T cell deletion. Although the critical molecular 
      mechanism of CP remains to be elucidated, accumulating evidence indicates that 
      exogenous antigens are processed through endoplasmic reticulum-associated 
      degradation (ERAD) after export from non-classical endocytic compartments. Until 
      recently, characterizations of these endocytic compartments were limited because 
      there were no specific molecular markers other than exogenous antigens. The 
      method described here is a new vesicle isolation protocol, which allows for the 
      purification of these endocytic compartments. Using this purified microsome, we 
      reconstituted the ERAD-like transport, ubiquitination, and processing of the 
      exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system 
      processed the exogenous antigen after export from this cellular compartment. This 
      protocol can be further applied to other cell types to clarify the molecular 
      mechanism of CP.
FAU - Imai, Jun
AU  - Imai J
AD  - Laboratory of Physiological Chemistry, Faculty of Pharmacy, Takasaki University 
      of Health and Welfare; jimai@takasaki-u.ac.jp.
FAU - Otani, Mayu
AU  - Otani M
AD  - Laboratory of Physiological Chemistry, Faculty of Pharmacy, Takasaki University 
      of Health and Welfare.
FAU - Sakai, Takahiro
AU  - Sakai T
AD  - Laboratory of Physiological Chemistry, Faculty of Pharmacy, Takasaki University 
      of Health and Welfare.
FAU - Hatta, Shinichi
AU  - Hatta S
AD  - Laboratory of Physiological Chemistry, Faculty of Pharmacy, Takasaki University 
      of Health and Welfare.
LA  - eng
PT  - Journal Article
PT  - Video-Audio Media
DEP - 20170821
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (Antigens)
RN  - 0 (Histocompatibility Antigens Class I)
SB  - IM
MH  - Antigen Presentation/*immunology
MH  - Antigens/*immunology
MH  - Cross-Priming/*genetics
MH  - Dendritic Cells/*immunology
MH  - Endoplasmic Reticulum-Associated Degradation/*genetics
MH  - Histocompatibility Antigens Class I/*immunology
MH  - Humans
PMC - PMC5614360
EDAT- 2017/09/06 06:00
MHDA- 2018/05/15 06:00
PMCR- 2019/08/21
CRDT- 2017/09/06 06:00
PHST- 2017/09/06 06:00 [entrez]
PHST- 2017/09/06 06:00 [pubmed]
PHST- 2018/05/15 06:00 [medline]
PHST- 2019/08/21 00:00 [pmc-release]
AID - 55949 [pii]
AID - 10.3791/55949 [doi]
PST - epublish
SO  - J Vis Exp. 2017 Aug 21;(126):55949. doi: 10.3791/55949.