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INNATE IMMUNITY 3
pSELECT-GFP-LC3 - GFP-LC3 Expression Plasmid
Description
pSELECT-GFP-LC3 is a mammalian expression vector containing the
human LC3B gene fused at its 5’ end to the green fluorescent protein
(GFP) gene. The same plasmid is available with the GFP gene alone as a
control. This control plasmid is called pSELECT-NGFP-zeo. Both plasmids
are selectable in bacteria and mammalian cells with Zeocin .
Application
Expression of the GFP-LC3 fusion gene allows to visualize autophagosome
formation in real time in live cells. During autophagosome formation, GFP-
LC3 is processed and recruited to the autophagosome membrane, where
it can be imaged as cytoplasmic puncta by fluorescence microscopy (see
picture). The percentage of GFP-LC3 positive cells can be determined and
is indicative of autophagosome formation.
Contents and Storage
pSELECT-GFP-LC3 and pSELECT-NGFP-zeo are provided as 20 μg of
lyophilized DNA. The plasmids can be purchased individually or together.
They are shipped at room temperature and should be stored at -20°C.
Both plasmids are provided with 4 pouches of E. coli Fast-Media ® Zeo (2
TB and 2 Agar). For more information about Fast-Media ® , see pages 44-45.
Autophagy Inducers & Inhibitors
Green puncta representing
autophagosome formation in cells
expressing a GFP-LC3 fusion.
PRODUCT QUANTITY CAT. CODE
pSELECT-GFP-LC3 20 μg psetz-gfplc3
pSELECT-NGFP-zeo 20 μg psetz-ngfp
Autophagy is regulated at different levels. Two connected signaling pathways encompassing class III phosphatidylinositol 3-kinase (PI3K) and
mammalian target of rapamycin (mTOR) play a central role in this regulation. Autophagy requires PI3K autophagy but is negatively regulated
by mTOR. Thus inhibitors of PI3K act as autophagy inhibitors while inhibitors of mTOR act as autophagy inducers.
PRODUCT DESCRIPTION
Autophagy Inducers
Rapamycin (mTOR inhibitor)
Tamoxifen
Autophagy Inhibitors
mimics cellular starvation by blocking signals required for
cell growth and proliferation
stimulates autophagy by increasing the intracellular level
of ceramide and abolishing the inhibitory effect of PI3K
1. Jung CH. et al., 2010. mTOR regulation of autophagy. FEBS Lett. 584(7):1287-95.
2. Scarlatti F. et al., 2004. Ceramide-mediated macroautophagy involves inhibition of
protein kinase B and up-regulation of beclin 1. J Biol Chem. 279(18):18384-91. 3.
Blommaart EF. et al., 1997.The phosphatidylinositol 3-kinase inhibitors wortmannin
and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur. J. Biochem. 243: 240–
246. 4. Yamamoto A. et al., 1998. Bafilomycin A1 prevents maturation of autophagic
vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat
hepatoma cell line, H-4-II-E cells. Cell Struct Funct. 23(1):33-42.
WORKING
CONCENTRATION QTY
CATALOG
CODE
10 - 500 nM 5 mg tlrl-rap 1
10 - 100 μM 200 mg tlrl-txf 2
3-Methyladenine (3-MA, PI3K inhibitor) inhibits the formation of autophagosome 5 mM 50 mg tlrl-3ma 3
Bafilomycin A1 (V-ATPase inhibitor) prevents maturation of autophagic vacuoles 0.1 - 1 μM 10 μg tlrl-baf 4
LY294002 (PI3K inhibitor) inhibits the formation of autophagosome 10 - 100 μM 5 mg tlrl-ly29 3
Wortmannin (PI3K inhibitor) inhibits the formation of autophagosome 0.1 - 10 μM 5 mg tlrl-wtm 3
Contents and Storage
102 www.invivogen.com/autophagy
REF.
Each product is provided as a solid and shipped at room temperature.
Store at room temperature, 4°C or -20°C according to the product label.