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KU-0063794

Name: KU-0063794
Brand: Selleck Chemicals
SKU: S1226
Rating: 5 (65 reviews)

Catalog No.S1226

For research use only.

KU-0063794 is a potent and highly specific dual-mTOR inhibitor of mTORC1 and mTORC2 with IC50 of ~10 nM in cell-free assays; no effect on PI3Ks.

CAS No. 938440-64-3

Selleck's KU-0063794 has been cited by 65 publications

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mTOR Signaling Pathway Map

Biological Activity

Description

KU-0063794 is a potent and highly specific dual-mTOR inhibitor of mTORC1 and mTORC2 with IC50 of ~10 nM in cell-free assays; no effect on PI3Ks.

Targets

mTORC1 ^[1] (Cell-free assay)	mTORC2 ^[1] (Cell-free assay)
~10 nM	~10 nM

In vitro

Compared with the mTOR inhibitor PP242, KU-0063794 exhibits higher specificity for mTOR, as being inactive against PI3Ks or 76 other kinases. In HEK-293 cells, KU-0063794 at 30 nM is sufficient to rapidly ablate S6K1 activity by blocking the phosphorylation of the hydrophobic motif (Thr³⁸⁹) and subsequently the phosphorylation of the T-loop residue (Thr²²⁹). In case of IGF1 stimulation of serum-starved HEK-293 cells, 300 nM of KU-0063794 is needed to inhibit the S6K1 activity by ~90%. KU-0063794 at 100-300 nM also completely inhibits the amino-acid-induced phosphorylation of S6K1 and S6 protein. Similar to S6K1, KU-0063794 inhibits the phosphorylation of mTORC1 at Ser²⁴⁴⁸ and mTORC2 at Ser²⁴⁸¹ in a dose-dependent and time-dependent manner. In the presence of serum or following IGF1 stimulation, KU-0063794 induces a dose-dependent inhibition of the activity and phosphorylation of Akt at Ser⁴⁷³ and unexpected Thr³⁰⁸ as well as the phosphorylation of the Akt substrates PRAS40 at Thr²⁴⁶, GSK3α/GSK3β at Ser²¹/Ser⁹ and Foxo-1/3a at Thr²⁴/Thr³². KU-0063794 but not rapamycin inhibits SGK1 activity and Ser⁴²² phosphorylation as well as its physiological substrate NDGR1 in a dose-dependent manner, to the same extent as S6K1 and Akt phosphorylation, whereas KU-0063794 dose not inhibit phorbol ester induced ERK or RSK phosphorylation and RSK activation. Compared with rapamycin, KU-0063794 exhibits more significant potency to induce the complete dephosphorylation of 4E-BP1 at Thr³⁷, Thr⁴⁶ and Ser⁶⁵. KU-0063794 inhibits cell growth of both wild-type and mLST8-deficient MEFs and induces a G1 cell cycle arrest, more significantly than rapamycin. ^[1]

Cell Data

Cell Lines	Assay Type	Concentration	Incubation Time	Formulation	Activity Description	PMID

HepG2	MUPD[YxtKF[rYXLpcIl1gSCDc4PhfS=>	MVuwMlHjiJN3MNMg{txO	NI\1Opc4OiCq		M2XIdoRm[3KnYYPld{Bk\WyuII\pZYJqdGm2eTDpckBiKGSxc3Wg[IVx\W6mZX70JI1idm6nch?=	NHHRZos9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{NkK3PFgyQSd-Mk[yO\|g5OTl:L3G+
HepG2	MX\Dc4xwdnliRn;ycYF1cW:wIFHzd4F6	MonvNgKBmzVywrFOwG0>	NEDpOXkyOCCm		M4LY[YRm[3KnYYPld{B1cGViboXtZoVzKG:oII\pZYJt\SCKZYDHNkBkd2yxbnnld:Khe2mpbnnmbYNidnSueR?=	NXPrZVBLRGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC\|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMk[yO\|g5OTlpPkK2Nlc5QDF7PD;hQi=>
HepG2	NVO0SnVFSXCxcITvd4l{KEG\|c3H5	NI[2VooxNjIkgKO1NOKh\|ryP	MmLlOFghcA>?		NWKx[HdRcW6mdXPld{BieG:ydH;zbZMhcW5iYTDkc5NmKGSncHXu[IVvfCCvYX7u[ZI>	MV:8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zPjJ5OEixPUc,OjZ{N{i4NVk9N2F-
HepG2	MVnGeY5kfGmxbjDBd5NigQ>?	MlXFOU8yOCEQvF2=	MnXlNlQhcA>?		M3jpfYRz[W2jdHnjZYxtgSCrbnjpZol1eyCyaH;zdIhwenmuYYTpc44hd2ZiQVvUJIF1KFOncj20O\|M>	NXrqdnhmRGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC\|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMk[yO\|g5OTlpPkK2Nlc5QDF7PD;hQi=>
HepG2	Ml\6SpVv[3Srb36gRZN{[Xl?	NVrtNlREPS9zMDFOwG0>	MX2yOEBp		M2TIdIRwf26{ZXf1cIF1\XNidHjlJIxmfmWuczDITWYy\|rFiYX7kJIN6[2yrbjDENeKh	MmLCQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOjZ{N{i4NVkoRjJ4Mke4PFE6RC:jPh?=
HepG2	NUnWW29KTnWwY4Tpc44hSXO\|YYm=	MVKwMlHjiJN3MNMg{txO	NYq4WXl3OjRiaB?=		MX\pcoR2[2W\|IIC2NkBld3ewcnXneYxifGmxbjygRoVkdGmwLUGg[ZhxemW\|c3nvckBidmRiTFOzRk1KKHSxIFzDN2IuUUliY3;ueoVze2mxbjDpckBiKGSxc3Wg[IVx\W6mZX70JI1idm6nch?=	NFnybpo9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{NkK3PFgyQSd-Mk[yO\|g5OTl:L3G+
HepG3	NXXiSnNPTnWwY4Tpc44hSXO\|YYm=	M4r5PVAvOeLCk{WwxsDPxE1?	MX20PEBp		MUXpcoR2[2W\|IHPlcIwh[XW2b4DoZYd6KGmwIHGg[I9{\SCmZYDlcoRmdnRibXHucoVz	NFTtO4E9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{NkK3PFgyQSd-Mk[yO\|g5OTl:L3G+
AGS	NFW2VXdIem:5dHigTY5pcWKrdHnvckBCe3OjeR?=				NYXCfXhEUUN3ME2xOU4xKMLzIEKuPVEh\|ryP	NFq3eoM9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{NEW5O\|Q4QCd-MkS1PVc1Pzh:L3G+
HGC27	M3e0Vmdzd3e2aDDJcohq[mm2aX;uJGF{e2G7				MYLJR\|UxRTF3LkCgxtEhPC56MjFOwG0>	M{TFXlxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ2NUm3OFc5Lz5{NEW5O\|Q4QDxxYU6=
MKN45	MlrMS5Jwf3SqIFnubIljcXSrb36gRZN{[Xl?				NInadVdKSzVyPUCuPFIhyrFiMD6wNUDPxE1?	MWG8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zPDV7N{S3PEc,OjR3OUe0O\|g9N2F-
NUGC4	NYG3bGo3T3Kxd4ToJGlvcGmkaYTpc44hSXO\|YYm=				MnPHTWM2OD1{LkmzJOKyKDBwM{Gg{txO	MVK8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zPDV7N{S3PEc,OjR3OUe0O\|g9N2F-
PC9	NUL2fnVTT3Kxd4ToJGlvcGmkaYTpc44hSXO\|YYm=		MlfhO\|IhcA>?		MX\JR\|UxRTFyLkG1xtExNjZ{IH7N	MXy8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zOzh5NEi4NEc,OjN6N{S4PFA9N2F-
PC9GR	NVjvNlMxT3Kxd4ToJGlvcGmkaYTpc44hSXO\|YYm=		NGXIZ2Y4OiCq		MYHJR\|UxRTZwMkJCtVEvOzBibl2=	Ml;HQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOjN6N{S4PFAoRjJ\|OEe0PFgxRC:jPh?=
H1650	Ml7tS5Jwf3SqIFnubIljcXSrb36gRZN{[Xl?		NXnxWmdMPzJiaB?=		NVPDeIwzUUN3ME23MlYyyrFyLk[yJI5O	NEiwb5M9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{M{i3OFg5OCd-MkO4O\|Q5QDB:L3G+
H1975	M2nSWmdzd3e2aDDJcohq[mm2aX;uJGF{e2G7		NYDUd2lRPzJiaB?=		NEDicFJKSzVyPUGxMlE2yrFyLkmzJI5O	M{TPN\|xiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ\|OEe0PFgxLz5{M{i3OFg5ODxxYU6=
PC9	MofHSpVv[3Srb36gRZN{[Xl?	NW\uVHBSOTBwMUWgcm0>	M2e4V\|czKGh?		NWPyOIs3cW6qaXLpeJMhdVSRUjDwbI9{eGixconsZZRqd25ic4TheJV{yqB?	NIDobnc9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{M{i3OFg5OCd-MkO4O\|Q5QDB:L3G+
PC9GR	Mn;xSpVv[3Srb36gRZN{[Xl?	NVPJR\|FlPi5{MTDuUS=>	MW[3NkBp		Mm\QbY5pcWKrdIOgcXRQWiCyaH;zdIhwenmuYYTpc44he3SjdIXzxsA>	NUXWSY9yRGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC\|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMkO4O\|Q5QDBpPkKzPFc1QDhyPD;hQi=>
H1650	MknsSpVv[3Srb36gRZN{[Xl?	NVyyVZgyPy54MTDuUS=>	MoT4O\|IhcA>?		NITHc5VqdmirYnn0d{BuXE:UIIDoc5NxcG:{eXzheIlwdiC\|dHH0eZPDqA>?	Mkf5QIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOjN6N{S4PFAoRjJ\|OEe0PFgxRC:jPh?=
H1975	MXXGeY5kfGmxbjDBd5NigQ>?	NWroUHQ{OTFwMUWgcm0>	M4P5S\|czKGh?		MmHSbY5pcWKrdIOgcXRQWiCyaH;zdIhwenmuYYTpc44he3SjdIXzxsA>	MXy8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zOzh5NEi4NEc,OjN6N{S4PFA9N2F-
PC9	MkS0SpVv[3Srb36gRZN{[Xl?	MkTxNVAvOTVibl2=	MWK3NkBp		NHvGSVJqdmirYnn0d{BxcG:\|cHjvdplt[XSrb36gc4YheDdyU{\L	MYq8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zOzh5NEi4NEc,OjN6N{S4PFA9N2F-
PC9GR	NHOxfotHfW6ldHnvckBCe3OjeR?=	NEXVfms3NjJzIH7N	MXm3NkBp		NF\EPY9qdmirYnn0d{BxcG:\|cHjvdplt[XSrb36gc4YheDdyU{\L	MXu8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zOzh5NEi4NEc,OjN6N{S4PFA9N2F-
H1650	MYHGeY5kfGmxbjDBd5NigQ>?	NEXROFU4NjZzIH7N	MlnCO\|IhcA>?		NInhZ4RqdmirYnn0d{BxcG:\|cHjvdplt[XSrb36gc4YheDdyU{\L	MlP5QIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOjN6N{S4PFAoRjJ\|OEe0PFgxRC:jPh?=
H1975	NHTjdYpHfW6ldHnvckBCe3OjeR?=	NXnjTGw1OTFwMUWgcm0>	M2fUW\|czKGh?		NX34cGlHcW6qaXLpeJMheGixc4Doc5J6dGG2aX;uJI9nKHB5MGO2Ty=>	MniwQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOjN6N{S4PFAoRjJ\|OEe0PFgxRC:jPh?=
LNCaP	MXTD[YxtKF[rYXLpcIl1gSCDc4PhfS=>	MYiwMVExKM7:TR?=	NUHnW4hIOjRiaNMg		Mn;N[IVkemWjc3XzJINmdGxidnnhZoltcXS7IHnuJIEh\G:\|ZTDk[ZBmdmSnboSgcYFvdmW{	NHHaT2o9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{M{i0NFYxPSd-MkO4OFA3ODV:L3G+
PC-3	MXLD[YxtKF[rYXLpcIl1gSCDc4PhfS=>	M3qxO\|AuOTBizszN	MXmyOEBpyqB?		NWnRXJRL\GWlcnXhd4V{KGOnbHygeoli[mmuaYT5JIlvKGFiZH;z[UBl\XCnbnTlcpQhdWGwbnXy	NHHjcXI9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{M{i0NFYxPSd-MkO4OFA3ODV:L3G+
MDA-MB-468	MX7D[YxtKF[rYXLpcIl1gSCDc4PhfS=>	NYLLO\|RkOC1zMDFOwG0>	MofDNlQhcMLi		M4DIToRm[3KnYYPld{Bk\WyuII\pZYJqdGm2eTDpckBiKGSxc3Wg[IVx\W6mZX70JI1idm6nch?=	MUW8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zOzh2ME[wOUc,OjN6NEC2NFU9N2F-
LNCaP	NHfGeJRHfW6ldHnvckBCe3OjeR?=	MXGyNFDjiJN6MECgcm0>	NWnmNlhwOjRiaNMg		MkHY[IVkemWjc3XzJJRp\SCyaH;zdIhwenmuYYTpc44hdGW4ZXygc4YheDdyU{\LJIlvKGFiZH;z[UBl\XCnbnTlcpQhdWGwbnXy	M3\NfFxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ\|OESwOlA2Lz5{M{i0NFYxPTxxYU6=
PC-3	M3HyXGZ2dmO2aX;uJGF{e2G7	MWqyNFDjiJN6MECgcm0>	NFHCcIozPCCqwrC=		NHPBb3Fl\WO{ZXHz[ZMhfGinIIDoc5NxcG:{eXzheIlwdiCuZY\lcEBw\iCyN{DTOmshcW5iYTDkc5NmKGSncHXu[IVvfCCvYX7u[ZI>	NYPKc5RlRGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC\|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMkO4OFA3ODVpPkKzPFQxPjB3PD;hQi=>
MDA-MB-468	Mnr4SpVv[3Srb36gRZN{[Xl?	M3j4T\|IxOOLCk{iwNEBvVQ>?	NH:4NI8zPCCqwrC=		MYLk[YNz\WG\|ZYOgeIhmKHCqb4PwbI9zgWyjdHnvckBt\X[nbDDv[kBxPzCVNlugbY4h[SCmb4PlJIRmeGWwZHXueEBu[W6wZYK=	MoPMQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOjN6NEC2NFUoRjJ\|OESwOlA2RC:jPh?=
Caki-1	NFzxbVdHfW6ldHnvckBCe3OjeR?=	NYPMRnE2OTByLUKwNFAhdk1?	MV:xNE0yQDBibXnu	M3fl[GROW09?	M2\JRYlvcGmkaYTzJIJwfGhibWTPVmMyKGGwZDDtWG9TSzJiYYOgbY5lcWOjdHXkJIJ6KHSqZTDk[YNz\WG\|ZTDpckBxcG:\|cHjvdplt[XSrb36gc4Yh\G:5boP0doVidSCnZn\lZ5RwenN?	MYi8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zOzN2OUm4PUc,OjN\|NEm5PFk9N2F-
786-O	Mm\6SpVv[3Srb36gRZN{[Xl?	MXqxNFAuOjByMDDuUS=>	MWixNE0yQDBibXnu	MYLEUXNQ	MYXpcohq[mm2czDic5RpKG2WT2LDNUBidmRibWTPVmMzKGG\|IHnu[Ilk[XSnZDDifUB1cGViZHXjdoVie2ViaX6gdIhwe3Cqb4L5cIF1cW:wIH;mJIRwf26\|dILlZY0h\W[oZXP0c5J{	NEjIR4E9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{M{O0PVk5QSd-MkOzOFk6QDl:L3G+
Caki-1	MUDD[YxtKF[rYXLpcIl1gSCDc4PhfS=>	NFK0eXA{ODBvNECwNEBvVQ>?	MVOyOE06PiCq	M3ns[2ROW09?	M1rISpN2eHC{ZYPz[ZMhfGinIHPlcIwhfmmjYnnsbZR6KGmwIHLveIghfGmvZTDhcoQh\G:\|ZTDk[ZBmdmSnboSgcYFvdmW{	M4nFZVxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ\|M{S5PVg6Lz5{M{O0PVk5QTxxYU6=
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Rh18	M2CzbJFJXFNiYYPzZZk>				NF;VVpNyUFSVIH;mJJBm\GmjdILpZ{Bk[W6lZYKgZ4VtdCCuaX7ld{B1dyCrZHXueIlngSCvdXz0bZBt\SCxcIDvdpR2dmm2aXXzJIZweiCmcoXnJJJmeHW{cH;zbY5oQiCFb37mbZJu[XSxcomgd4Nz\WWwIH\vdkBTcDF6IHPlcIx{	M33PSlxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ7NEO1NVM6Lz5{OUSzOVE{QTxxYU6=
Rh30	M37YSZFJXFNiYYPzZZk>				NGntUFJyUFSVIH;mJJBm\GmjdILpZ{Bk[W6lZYKgZ4VtdCCuaX7ld{B1dyCrZHXueIlngSCvdXz0bZBt\SCxcIDvdpR2dmm2aXXzJIZweiCmcoXnJJJmeHW{cH;zbY5oQiCFb37mbZJu[XSxcomgd4Nz\WWwIH\vdkBTcDNyIHPlcIx{	NHL0NVU9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{OUSzOVE{QSd-Mkm0N\|UyOzl:L3G+
SK-N-SH	NFXJd2RyUFSVIHHzd4F6				MWnxTHRUKG:oIIDl[IlifHKrYzDjZY5k\XJiY3XscEBtcW6nczD0c{Bq\GWwdHnmfUBufWy2aYDs[UBweHCxcoT1col1cWW\|IH\vdkBlenWpIILldJVzeG:\|aX7nPkBEd26oaYLtZZRwenlic3Py[YVvKG[xcjDTT{1PNVOKIHPlcIx{	MYW8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zQTR\|NUGzPUc,Ojl2M{WxN\|k9N2F-

Click to View More Cell Line Experimental Data

Assay

Methods	Test Index	PMID

Western blot

p-S6K / S6K / p-4E-BP1 / E7 / E6 / p53 ; p-mTOR

28115701 24262658

In vivo

Ku0063794 inhibits tumor growth and mTOR signaling in a preclinical renal cell carcinoma model. However, Ku0063794 was not more effective than temsirolimus in the animal study. A possible explanation for lack of greater activity in vivo for Ku0063794 is that temsirolimus has important effects on the tumor microenvironment. Temsirolimus decreased angiogenesis in the xenograft tumors while Ku0063794 did not. Temsirolimus treated tumors expressed less VEGF and PDGF than Ku0063794 treated tumors, thus stimulating less angiogenesis^[2].

Protocol (from reference)

Kinase Assay: ^[1]	mTOR complexes kinase assays: HEK-293 cells are freshly lysed in Hepes lysis buffer. Lysate (1-4 mg) is pre-cleared by incubating with 5-20 μL of Protein G-Sepharose conjugated to pre-immune IgG. The lysate extracts are then incubated with 5-20 μL of Protein G-Sepharose conjugated to 5-20 μg of either anti-Rictor or anti-Raptor antibody, or pre-immune IgG. All antibodies are covalently conjugated to Protein G-Sepharose. Immunoprecipitations are carried out for 1 hour at 4 °C on a vibrating platform. The immunoprecipitates are washed four times with Hepes lysis buffer, followed by two washes with Hepes kinase buffer. For Raptor immunoprecipitates used for phosphorylating S6K1, for the initial two wash steps the buffer includes 0.5 M NaCl to ensure optimal kinase activity. GST-Akt1 is isolated from serum-deprived HEK-293 cells incubated with PI-103 (1 μM for 1 hour). GST-S6K1 is purified from serum-deprived HEK-293 cells incubated with rapamycin (0.1 μM for 1 hour). mTOR reactions are initiated by adding 0.1 mM ATP and 10 mM MgCl₂ in the presence of various concentrations of KU-0063794 and GST-Akt1 (0.5 μg) or GST-S6K1 (0.5 μg). Reaction are carried out for 30 minutes at 30 °C on a vibrating platform and stopped by addition of SDS sample buffer. Reaction mixtures are then filtered through a 0.22-μm-poresize Spin-X filter and samples are subjected to electrophoresis and immunoblot analysis with the indicated antibodies.
Cell Research: ^[1]	Cell lines: Wild-type and mLST8 deficient MEFs Concentrations: Dissolved in DMSO, final concentration ~3 μM Incubation Time: 24, 48, and 72 hours Method: Cells are treated with KU-0063794 for 24, 48, and 72 hours, and the medium is changed every 24 hours with freshly dissolved KU-0063794. For the measurement of cell growth, cells are washed once with PBS, and fixed in 4% (v/v) paraformaldehyde in PBS for 15 minutes. After washing once with water, the cells are stained with 0.1% Crystal Violet in 10% ethanol for 20 minutes and washed three times with water. Crystal Violet is extracted from cells with 0.5 mL of 10% (v/v) ethanoic (acetic) acid for 20 minutes. The eluate is then diluted 1:10 in water and absorbance at 590 nm is quantified. For the assessment of cell cycle distribution, cells are harvested by trypsinization, washed once in PBS, and re-suspended in ice-cold aq. 70% (v/v) ethanol. Cells are washed twice in PBS plus 1% (w/v) BSA and stained for 20 minutes in PBS plus 0.1% (v/v) Triton X-100 containing 50 g/mL propidium iodide and 50 g/mL RNase A. The DNA content of cells is determined using a FACSCalibur flow cytometer and CellQuest software. Red fluorescence (585 nm) is acquired on a linear scale, and pulse width analysis is used to exclude doublets. Cell-cycle distribution is determined using FlowJo software.
Animal Research: [2]	Animal Models: Nu/Nu nude mice Dosages: 8 mg/kg Administration: i.p.

References

Solubility (25°C)

In vitro


In vivo	Add solvents to the product individually and in order (Data is from Selleck tests instead of citations): 30% PEG400+0.5% Tween80+5% propylene glycol For best results, use promptly after mixing. Click to purchase: PEG400 Tween 80	30 mg/mL

Chemical Information

Molecular Weight	465.54
Formula	C₂₅H₃₁N₅O₄
CAS No.	938440-64-3
Storage	3 years	-20°C	powder
Storage	2 years	-80°C	in solvent
Smiles	CC1CN(CC(O1)C)C2=NC3=C(C=CC(=N3)C4=CC(=C(C=C4)OC)CO)C(=N2)N5CCOCC5

Download KU-0063794 SDF

In vivo Formulation Calculator (Clear solution)

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Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

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Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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