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NOVUS Biologicals Autophagy Total Solution - NO.1 NOVUS 2017-01-04 16:42:38

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Types of Autophagy
There are three main types of autophagy in mammalian cells: macroautophagy, microautophagy and chaperonemediated autophagy.
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Macroautophagy
Macroautophagy involves the creation of a phagophore, leading to the formation of the autophagosome which can consume whole organelles and deliver them to the lysosome for degradation. Different from microautophagy, double-membraned structures called autophagosomes enclose cellular material and then fuse with lysosomes.
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LC3
LC3 is a mammalian homolog of the yeast autophagy protein ATG8, originally identifid as microtubule associated protein1 light chain 3 (MAPLC3). LC3 is expressed as three splice variants (LC3A, LC3B and LC3C). Each of these splice variants exhibits different tissue distributions and is processed into two different post-translationally modifid forms, LC3-I and LC3-II. LC3-I is found in the cytosol while LC3-II localizes to autophagasome membranes. LC3-II is the fist mammalian protein identifid that specifially associates with the autophagosome membranes. In addition to acting as a marker for autophagosomes, the conversion of LC3-I to LC3-II can be used to demonstrate the induction of autophagy.
LC3B, NB600-1384 (Novus)
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Product 
Type

Target
Catalog#
Species
Clonality
Applications
Antibodies
LC3
NB100-2331
H, M, R +
Poly
WB, SW, FC, ICC, IHC, IP
NBP1-19167
H, M, R +
Poly
WB, SW, FC, ICC, IHC
NBP1-78964
H, M, R +
Poly
WB, ICC, IHC
NBP2-24392
H, M, R +
Poly
WB, IHC
LC3B
NB100-2220
H, M, R +
Poly
WB, SW, ICC, IHC, IP
NB600-1384
H, M, R +
Poly
WB, SW, EM, FC, ICC, IHC
LC3C
NBP2-36664
H
Poly
WB, ICC, IHC
NB110-74806
H
Poly
WB, IHC
LC3 
Antibody Pack

NB910-40435
H, M
Poly
WB, IHC
Proteins and
Peptides

LC3
NB100-2220PEP
 
 
 
Cell Lysates
LC3B
NBL1-12844
H
 
WB
LC3
NBP2-04906
H
 
WB
LC3
NBL1-12843
H
 
WB

 
Microautophagy
Microautophagy involves the sequestering of cytosolic components at the surface of the lysosome. Compared to macroautophagy, microautophagy differs only in that the lysosome or vacuole sequesters proteins for degradation directly on their membrane surface. Therefore there are no autophagosome transport vesicles in microautophagy.
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Chaperone-Mediated Autophagy
Chaperone-mediated autophagy (CMA) involves the chaperone-dependent selection of soluble cytosolic proteins which are then targeted to lysosomes, before directly translocated across the lysosome membrane for degradation. The unique features of chaperone-mediated autophagy are the selectivity on the proteins that are degraded by this pathway and the direct shuttling of these proteins across the lysosomal membrane without the requirement for the formation of additional vesicles.
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Components of Chaperone-Mediated Autophagy
Components 
of CMA

Antibody 
Target

Cat#
Species
Clonality
Applications
Substrates
Aldolase B
NBP2-15345
H, M, R
Poly
WB, ICC, IHC
Eps8
NBP2-27300
H
Mono
WB, IHC
Pax2
NB600-1455
H
Poly
WB, ICC, IHC
MEF2D
NBP2-17261
H
Poly
WB
RCAN1
NBP1-46852
H, M
Poly
WB, IHC
a-synuclein
NBP1-26380
H, M
Mono
WB, FC, ICC, IHC
Chaperones
Hsp70
NBP1-77455
H, M
Poly
SW, FC, ICC, IHC
Hsp90 alpha
NBP1-77682
H, M
Poly
ICC, IHC
Hsp90 beta
NBP1-77561
H, M, R
Poly
ICC, IHC
Receptor
LAMP2A
NBP2-22217
H
Mono
WB, SW, FC, ICC, IHC
Regulators
EF1 alpha
NBP1-55245
H, M, P +
Poly
WB

 
Subcellular Structure-Specifi Autophagy
Depending on different subcellular structures that are specifially targeted for lysosomal degradation, autophagy processes include: mitophagy, ribophagy, lipophagy, pexophagy, aggrephagy and xenophagy.
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Mitophagy
Mitophagy is a process by which mitochondria are targeted for degradation via autophagy pathway. Mitophagy is mediated by ATG32 (in yeast) and NIP3-like protein X (NIX). Mitophagy is also regulated by PINK1 and Parkin protein.
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Ribophagy
Ribophagy is a type of macroautophagy that selectively degrades ribosomes or ribosome-RNA complexes. Doublemembraned autophagosomes enclose ribosomes, and then fuse with lysosomes for degradation.
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Aggrephagy
Protein aggregation is a continuous process in cells. Proteins that are damaged beyond repair can be degraded by the lysosome via autophagy. The selective degradation of protein aggregates by macroautophagy is called aggrephagy.
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Autophagy Processes
Autophagy processes include autophagy initiation, autophagosome formation, autolysosome fusion, degradation and recycling. Major protein complexes involved in these processes include ATG1/ULK complex, PI 3 K complex, ATG8/Ubl conjugation, ATG12 Ubl conjugation, and ATG9 cycling system.
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Autophagy Initiation
Autophagy initiation involves ATG1/ULK complex, PI3K complex, LC3 recruitment and p62 binding
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ATG1/ULK Complex
Target
Cat#
Species
Clonality
Applications
ULK1
NBP2-24738
H, M, Pr
Poly
WB, IHC
ULK2
NBP1-33136
H, M
Poly
WB, ICC, IHC
Phospho-
ATG13 (S318)

NBP2-19127
H
Poly
WB, ELISA
FIP200
NB100-77279
H, M
Poly
WB, IP
ATG101
NBP1-88877
H
Poly
WB, IHC

 
PI3K Complex
 
Beclin 1 and Beclin 2
 
Beclin 1 is a subunit of the Class III PI 3 K complex. The binding of Beclin 1 to the pre-autophagosomal structure initiates the formation of the autophagosome and is therefore required for autophagy. Beclin 1 was the fist mammalian gene to be identifid that mediates autophagy; it also has tumor suppressor and antiviral functions. Beclin 2 is a novel coiled-coil protein related to Beclin 1. It is thought to interact with Bcl-2, an anti-apoptotic protein, and is believed to function in autophagy.
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Antibody Target
Cat#
Species
Clonality
Applications
ATG14L
NBP2-36445
H, M, R
Poly
WB, ELISA, ICC
VPS34
NB110-87320
H, M, R +
Poly
WB, SW, ICC
PIK3R4 (VPS15)
NBP1-30463
H, M
Poly
WB, ICC, IP
Ambra1
NBP1-07124
H, M, R
Poly
WB, ELISA, ICC, IHC
Beclin 1
NB500-249
H, M, R
Poly
WB, SW, ICC, IHC, IP
Beclin 2
NB110-60984
H
Poly
WB, SW

 
Autophagosome Formation
The formation of autophagosomes involves ATG12 Ubiquitin-like (Ubl) conjugation and LC3/ATG8 conjugation.
LC3/ATG8 Ubl conjugation
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Antibody Target
Cat#
Species
Clonality
Applications
p62/ SQSTM1
NBP1-42821
H, M, R
Poly
WB, ICC
LC3
NB100-2220
H, M, R +
Poly
WB, SW, ICC, IHC, IP
GABARAP
NBP1-97416
M
Poly
IHC
ATG4B
NBP2-24709
H, M, R
Poly
WB, IHC
ATG7
NBP1-95872
H, M, R
Mono
WB, ICC
LC3B
NB600-1384
H, M, R +
Poly
WB, SW, EM, FC, ICC, IHC

 
ATG12 Ubl conjugation
ATG5 complexes with ATG12 and is required for the formation of the autophagosome. ATG5 is heavily expressed in dead tumor cells, making it a marker for successful anti-cancer therapies. The ATG12-ATG5-ATG16L complex is essential for the elongation of autophagic isolation membranes.
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Antibody
Cat#
Species
Clonality
Applications
ATG5
NB110-53818
H, M, R +
Poly
WB, SW, ICC, IHC, IP
ATG12
NBP2-15501
H, M, R
Poly
WB, ICC, IHC
ATG16L1
NB110-60928
H, M, R +
Poly
WB, EM, IHC

 
Autolysosome Fusion
The direct fusion of autophagosomes with lysosomes produces autolysosomes. The proteins involved in this process nclude LAMP1, Rab7, UVRAG and Rubicon.
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Degradation and Recycling
 
ATG9 cycling system
 
ATG9 is the only integral membrane protein required for autophagosome formation, and is considered a membrane carrier in autophagy-related pathways. It is regulated via ATG1 and is found migrating between mitochondria and the pre-autophagosomal structure.
 
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Regulation of Autophagy
 
Transcriptional regulation of autophagosome marker LC3
 
As an autophagosome marker, LC3 is considered the only well-characterized protein that is specifially localized to autophagic structures, throughout the process from phagophore to lysosomal degradation. At the transcriptional level, there are several factors that regulate (either up-regulate or down-regulate) the expression of LC3.
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Transcription
Factor

Regulation 
of LC3

Autophagy 
Effect

Cat#
Species
Clonality
Applications
ATF4
Up-regulation
Increased
NB100-81802
H
Poly
WB, IHC
CHOP
Up-regulation
Increased
NB600-1335
H, M, R +
Mono
WB, SW, GS,
ICC, IHC, IP

E2F1
Up-regulation
Increased
NB600-210
H, M
Mono
WB, ICC,
IHC, IP

FOXO1
Up-regulation
Increased
NBP2-31376
H
Mono
WB, SW,
ICC, IHC

JUN
Up-regulation
Increased
NB110-55569
H, M, R
Mono
WB, FC,
ICC, IHC, IP

TFEB
Up-regulation
Increased
NB100-1030
H
Poly
WB, IHC, ELISA
NBP2-12758
H
Poly
WB, IP
ZKSCAN3
Down-regulation
Decreased
NBP1-31566
H
Poly
WB, ICC, IHC

 
Protein Regulators
In addition to the transcriptional regulation of autophagy via LC3, there are many other proteins such as mTOR that regulate autophagy pathways.
 
mTOR
mTOR (mammalian Target of Rapamycin) is an evolutionarily-conserved protein kinase. As a central regulator of cell growth, mTOR plays a key role at the interface of the pathways that coordinate regulation of the balance between cell growth and autophagy in response to nutritional status, growth factor and stress signals.
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Negative Regulators of Autophagy
Target
Cat#
Species
Clonality
Applications
S6K
NBP2-23649
H, M, R +
Poly
WB, ELISA
p38
NB100-56665
H
Poly
WB, IHC
 
Positive Regulators of Autophagy
Target
Cat#
Species
Clonality
Applications
Beclin 1
NB110-87318
H, M, Ca +
Poly
WB, SW, ICC, IHC, IP
NB500-266
H, M
Poly
WB, ICC
VPS34 
(Class III PI3K)

NB110-87320
H, M, R +
Poly
WB, SW, ICC
ERK1
NB100-56376
H
Poly
WB, IHC
TRAIL
NB100-56518
H
Mono
WB, IHC
JNK2
NB600-1297
H
Poly
WB
BNIP3
NB100-56150
H, R, Ch +
Poly
WB, IHC, IP
TFEB
NB100-1030
H
Poly
WB, IHC, ELISA
NB100-93447
H, M, R +
Poly
WB, ELISA

 
Physiology and Pathology
 
Basal Autophagy and Induced Autophagy
 
Autophagy has many essential functions in cells and tissues. Basal autophagy is essential to remove damaged proteins and organelles, reduce ER stress, and limit the production of reactive oxygen species (ROS). Induced autophagy is important to provide nutrients and building blocks during periods of starvation. Autophagy is essential during the development and differentiation of many cell types, as well as in maintaining tissue homeostasis. Autophagy also plays an essential role during immunity. It is not surprising that dysregulation of autophagy has been implicated in the pathology of many diseases including cancer.
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Autophagy in Immunity
Autophagy is well integrated into immune regulation systems. Autophagy facilitates the recognition of infected cells by innate immune effectors. For example, although ATG3, ATG5 and ATG7 were found dispensable to be for the development of thymocytes, their absence impairs the survival and proliferation of peripheral T cells.
ATG5,NB110-53818 (Novus)
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Immune 
Functions

Target
Cat#
Species
Clonality
 Applications
Elimination of
microorganisms

SQSTM1
NBP1-48320
H, M, R
Poly
WB, SW, ICC, IHC
NOD2
NB100-524
H, M
Mono
WB, ICC, IHC, IP
ATG16L1
NB110-60928
H, M, R +
Poly
WB, EM, IHC
Control of proinflmmatory 
signaling

ATG5
NB110-53818
H, M, R +
Poly
WB, SW, ICC, IHC, IP
ATG12
NBP2-15501
H, M, R
Poly
WB, ICC, IHC
NLRP3
NBP2-12446
H, M, R +
Poly
WB, IHC
DICER
NB200-591
H, M
Poly
WB, IHC
Regulation of
adaptive immunity

IL-1β
NB600-633
H, M, Ba +
Poly
WB, ELISA, EM, IHC, IP
Secretion of
immune mediators

HMGB1
NB100-2322
H, M, R +
Poly
WB, SW, FC, ICC, IHC
NOX2
NBP1-41012
H
Mono
WB, ELISA, FC, ICC, IHC
TRAF6
NB100-56436
H, M
Poly
WB

 
Autophagy and Cancer
The role of autophagy in cancer is complex. During tumor initiation, autophagy acts as a barrier to cell transformation by reducing cell proliferation and DNA damage. However, during tumor progression, high levels of autophagy increase cancer cell survival. Cancers subsequently become dependent on autophagy to sustain cell growth.
 
Autophagy Proteins as Tumor Suppressors
Several lines of evidence have supported the view of autophagy as a novel mechanism of tumor suppression. Several autophagy proteins such as Beclin 1, Bif-1 and UVRAG have been found to function as tumor suppressors. Many anticancer agents such as tamoxifen and rapamycin, acting as potent inducers of autophagy, further support the prevailing view of autophagy as a mechanism of tumor suppression.
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Biochemical Analysis of Autophagy
Type
Name
Cat#
Peptides
ATG5 peptide
NB110-53818PEP
ATG16L1
NB110-60928PEP
ATG9A
NB110-56893PEP
Beclin 1
NB500-249PEP
LC3B
NB100-2220PEP
p62/SQSTM1
NBP1-42821PEP

 
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