TY - CHAP
T1 - Methods to study PTEN in mitochondria and endoplasmic reticulum
AU - Missiroli, Sonia
AU - Morganti, Claudia
AU - Giorgi, Carlotta
AU - Pinton, Paolo
N1 - Publisher Copyright:
© Springer Science+Business Media New York 2016.
PY - 2016
Y1 - 2016
N2 - Although PTEN has been widely described as a nuclear and cytosolic protein, in the last 2 years, alternative organelles, such as the endoplasmic reticulum (ER), pure mitochondria, and mitochondria-associated membranes (MAMs), have been recognized as pivotal targets of PTEN activity. Here, we describe different methods that have been used to highlight PTEN subcellular localization. First, a protocol to extract nuclear and cytosolic fractions has been described to assess the “canonical” PTEN localization. Moreover, we describe a protocol for mitochondria isolation with proteinase K (PK) to further discriminate whether PTEN associates with the outer mitochondrial membrane (OMM) or resides within the mitochondria. Finally, we focus our attention on a subcellular fractionation protocol of cells that permits the isolation of MAMs containing unique regions of ER membranes attached to the outer mitochondrial membrane (OMM) and mitochondria without contamination from other organelles. In addition to biochemical fractionations, immunostaining can be used to determine the subcellular localization of proteins; thus, a detailed protocol to obtain good immunofluorescence (IF) is described. The employment of these methodological approaches could facilitate the identification of different PTEN localizations in several physiopathological contexts.
AB - Although PTEN has been widely described as a nuclear and cytosolic protein, in the last 2 years, alternative organelles, such as the endoplasmic reticulum (ER), pure mitochondria, and mitochondria-associated membranes (MAMs), have been recognized as pivotal targets of PTEN activity. Here, we describe different methods that have been used to highlight PTEN subcellular localization. First, a protocol to extract nuclear and cytosolic fractions has been described to assess the “canonical” PTEN localization. Moreover, we describe a protocol for mitochondria isolation with proteinase K (PK) to further discriminate whether PTEN associates with the outer mitochondrial membrane (OMM) or resides within the mitochondria. Finally, we focus our attention on a subcellular fractionation protocol of cells that permits the isolation of MAMs containing unique regions of ER membranes attached to the outer mitochondrial membrane (OMM) and mitochondria without contamination from other organelles. In addition to biochemical fractionations, immunostaining can be used to determine the subcellular localization of proteins; thus, a detailed protocol to obtain good immunofluorescence (IF) is described. The employment of these methodological approaches could facilitate the identification of different PTEN localizations in several physiopathological contexts.
KW - Endoplasmic reticulum
KW - Immunofluorescence
KW - Mitochondria
KW - Mitochondria-associated membranes (MAMs)
KW - Nuclear extraction
KW - PTEN
KW - Subcellular fractionation
UR - http://www.scopus.com/inward/record.url?scp=84990194144&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84990194144&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-3299-3_13
DO - 10.1007/978-1-4939-3299-3_13
M3 - Chapter
C2 - 27033078
AN - SCOPUS:84990194144
T3 - Methods in Molecular Biology
SP - 187
EP - 212
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -