KU-55933 is a potent ATM inhibitor with an IC₅₀ and K_i of 12.9 and 2.2 nM, respectively, and is highly selective for ATM as compared to DNA-PK, PI3K/PI4K, ATR and mTOR.

KU-55933 (10 μM) blocks the ionizing radiation-induced p53 serine 15 phosphorylation. KU-55933 has a dose-dependent effect in inhibiting this ATM-dependent phosphorylation event with an estimated IC₅₀ of 300 nM. KU-55933 ablates the ionizing radiation-induced phosphorylation of these ATM substrates. KU-55933 specifically inhibits ATM but not the other DNA damage-activated PIKKs, ATR, and DNA-PK^[1]. KU-55933 induces pATM, p53, E2F1 and pATR, noticeably upregulates the nuclear fraction of E2F1 at the 0.5 h time point^[2]. Metformin increases ATM and AMPK phosphorylation, as well as SHP protein level in primary hepatocytes, and this stimulatory effect of metformin is repressed by a specific ATM kinase inhibitor KU-55933^[3].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

395.49

C₂₁H₁₇NO₃S₂

587871-26-9

Room temperature in continental US; may vary elsewhere.

DMSO : 80 mg/mL (202.28 mM; Need ultrasonic)

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.5 mg/mL (6.32 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (6.32 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.5 mg/mL (6.32 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (6.32 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 3.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.5 mg/mL (6.32 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (6.32 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Hickson I, et al. Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 2004 Dec 15;64(24):9152-9

  [2]. Khalil HS, et al. Pharmacological inhibition of ATM by KU55933 stimulates ATM transcription.Exp Biol Med (Maywood). 2012 Jun;237(6):622-34. Epub 2012 Jun 22.

  [3]. Kim YD, et al. Orphan nuclear receptor SHP negatively regulates growth hormone-mediated induction of hepatic gluconeogenesis through inhibition of STAT5 transactivation.J Biol Chem. 2012 Sep 12.

ATM for use in the in vitro assay is obtained by immunoprecipitation with rabbit polyclonal antiserum raised to the COOH-terminal 400 amino acids of ATM in buffer containing 25 mM HEPES (pH 7.4), 2 mM MgCl₂, 250 mM KCl, 500 μM EDTA, 100 μM Na₃VO₄, 10% v/v glycerol, and 0.1% v/v Igepal. ATM-antibody complexes are isolated from nuclear extract by incubating with protein A-Sepharose beads for 1 hour and then through centrifugation to recover the beads. In the well of a 96-well plate, ATM-containing Sepharose beads are incubated with 1 μg of substrate glutathione S-transferase-p53N66 (NH₂-terminal 66 amino acids of p53 fused to glutathione S-transferase) in ATM assay buffer [25 mM HEPES (pH 7.4), 75 mM NaCl, 3 mM MgCl₂, 2 mM MnCl₂, 50 μM Na₃VO₄, 500 μM DTT, and 5% v/v glycerol] at 37°C in the presence or absence of inhibitor. After 10 minutes with gentle shaking, ATP is added to a final concentration of 50 μM and the reaction continued at 37°C for an additional 1 hour. The plate is centrifuged at 250×g for 10 minutes (4°C) to remove the ATM-containing beads, and the supernatant is removed and transferred to a white opaque 96-well plate and incubated at room temperature for 1.5 hours to allow glutathione S-transferase-p53N66 binding. This plate is then washed with PBS, blotted dry, and analyzed by a standard ELISA technique with a phospho-serine 15 p53 antibody. The detection of phosphorylated glutathione S-transferase-p53N66 substrate is performed in combination with a goat antimouse horseradish peroxidase-conjugated secondary antibody. Enhanced chemiluminescence solutionis used to produce a signal and chemiluminescent detection is carried out via a TopCount plate reader.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

1BR or AT4 cells are seeded in 10-cm Petri dishes and treated on day 2 (80 to 90% confluence). Cells are preincubated for 1 hour with KU-55933 or vehicle control and then exposed to 5 Gy of ionizing radiation. Time courses of cell cycle distribution are performed, and the optimal time for discrimination of populations is selected as 16 hours. All subsequent experiments are performed at the 16-hour time point. Cells are stained with propidium iodide according to standard protocols and analyzed by FACS with a FACScalibur. Exponentially growing (50-70% confluent) SW620 cells in 60 mm dishes are exposed to KU-55933 or DMSO for 1 h before addition of etoposide (final concentration of 0.1 and 1 μM) for 16 h before harvesting, propidium iodide staining and analysis as above.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Hickson I, et al. Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 2004 Dec 15;64(24):9152-9

  [2]. Khalil HS, et al. Pharmacological inhibition of ATM by KU55933 stimulates ATM transcription.Exp Biol Med (Maywood). 2012 Jun;237(6):622-34. Epub 2012 Jun 22.

  [3]. Kim YD, et al. Orphan nuclear receptor SHP negatively regulates growth hormone-mediated induction of hepatic gluconeogenesis through inhibition of STAT5 transactivation.J Biol Chem. 2012 Sep 12.

KU-57788 (NU7441) is a highly potent and selective DNA-PK inhibitor with an IC₅₀ of 14 nM. KU-57788 is an NHEJ pathway inhibitor. KU-57788 also inhibits PI3K and mTOR with IC₅₀s of 5.0 and 1.7 μM, respectively^[1].

NU7441 (0.5 to 10 µM) inhibits the growth of liver cancer HepG2 cells dose- and time-dependently. NU7441 reduces pDNA-PKcs (S2056) protein expression in liver cancer cells. Furthermore, double treatment of NU7441 and 60Coγ IR affects DNA damage repair^[2]. NU7441 is solvent-exposed in BRD4, this inhibitor can be classified as a Type I BRD inhibitor^[4]. NU7441 reduces the frequency of NHEJ while increasing the rate of HDR following Cas9-mediated DNA cleavage^[5].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

KU-57788 (0.5 to 10 µM) inhibits the growth of liver cancer HepG2 cells dose- and time-dependently. KU-57788 reduces pDNA-PKcs (S2056) protein expression in liver cancer cells. Furthermore, double treatment of KU-57788 and 60Coγ IR affects DNA damage repair^[2]. KU-57788 weakly inhibits BRD4 and BRDT with IC₅₀s of 1 μM and 3.5 μM, respectively. KU-57788 is solvent-exposed in BRD4, this inhibitor can be classified as a Type I BRD inhibitor^[4]. KU-57788 reduces the frequency of NHEJ while increasing the rate of HDR following Cas9-mediated DNA cleavage^[5].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

413.49

C₂₅H₁₉NO₃S

503468-95-9

Room temperature in continental US; may vary elsewhere.

DMSO : 14.29 mg/mL (34.56 mM; Need ultrasonic)

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 1.43 mg/mL (3.46 mM); Clear solution

  此方案可获得 ≥ 1.43 mg/mL (3.46 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 14.3 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 1.43 mg/mL (3.46 mM); Clear solution

  此方案可获得 ≥ 1.43 mg/mL (3.46 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 14.3 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 3.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 1.43 mg/mL (3.46 mM); Clear solution

  此方案可获得 ≥ 1.43 mg/mL (3.46 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 14.3 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Justin J J Leahy, et al. Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. Bioorg Med Chem Lett. 2004 Dec 20;14(24):6083-7.

  [2]. Yang C, et al. NU7441 Enhances the Radiosensitivity of Liver Cancer Cells. Cell Physiol Biochem. 2016;38(5):1897-905

  [3]. Hardcastle IR, et al. Discovery of potent chromen-4-one inhibitors of the DNA-dependent protein kinase (DNA-PK) using a small-molecule library approach. J Med Chem. 2005 Dec 1;48(24):7829-46

  [4]. Ember SW, et al. The acetyl-lysine binding site of bromodomain-containing protein 4 (BRD4) interacts with diverse kinase inhibitors. ACS Chem Biol. 2014 Feb 25.

  [5]. Robert F, et al. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing. Genome Med. 2015 Aug 27;7:93

HepG2 cells (4000 per well) are cultured in a 96-well plate for 24 h. Once the cells complete the attachment, 0.1 µM, 1 µM, 5 µM, and 10 µM of KU-57788 are added to the culture media. After 12 h of KU-57788 treatment, 10% CCK-8 solution is added into the culture media, and the incubation continued for two h. OD450 values are determined by a spectrometer, and the results are analyzed to measure the cell growth.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Justin J J Leahy, et al. Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. Bioorg Med Chem Lett. 2004 Dec 20;14(24):6083-7.

  [2]. Yang C, et al. NU7441 Enhances the Radiosensitivity of Liver Cancer Cells. Cell Physiol Biochem. 2016;38(5):1897-905

  [3]. Hardcastle IR, et al. Discovery of potent chromen-4-one inhibitors of the DNA-dependent protein kinase (DNA-PK) using a small-molecule library approach. J Med Chem. 2005 Dec 1;48(24):7829-46

  [4]. Ember SW, et al. The acetyl-lysine binding site of bromodomain-containing protein 4 (BRD4) interacts with diverse kinase inhibitors. ACS Chem Biol. 2014 Feb 25.

  [5]. Robert F, et al. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing. Genome Med. 2015 Aug 27;7:93

KU-60019 is an improved ATM kinase-specific inhibitor with IC₅₀ of 6.3 nM.

KU-60019 is an improved analogue of KU-55933. KU-55933 has an IC₅₀ of 13 nM and K_i of 2.2 nM in vitro and is highly specific for the ATM kinase using a panel of 60 protein kinases. KU-60019 is an improved inhibitor of the ATM kinase with an IC₅₀ of 6.3 nM, approximately half that of KU-55933. The IC₅₀ values for DNA-PKcs and ATR are 1.7 and >10 μM, respectively, almost 270-and 1600-fold higher than for ATM. KU-60019 is 10-fold more effective than KU-55933 at blocking radiation-induced phosphorylation of key ATM targets in human glioma cells. In human U87 glioma cells, KU-55933 completely inhibits phosphorylation of p53 (S15) at 10 μM but not at 3 μM, whereas γ-H2AX levels are only partly reduced with 10 μM 1 h after irradiation. By comparison, 3 μM KU-60019 completely inhibits p53 phosphorylation and partial inhibits at 1 μM^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Despite PTEN-deficient control tumors reaching a 4-fold increase in size before PTEN wild-type controls, KU-60019-treated PTEN-deficient tumors display a statistically significant slowing in growth. This growth inhibition is especially evident at the start of the experiment (days 5-12) just after KU-60019 is administered (days 1-5)^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

547.67

C₃₀H₃₃N₃O₅S

925701-46-8

Room temperature in continental US; may vary elsewhere.

DMSO : 100 mg/mL (182.59 mM; Need ultrasonic)

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.5 mg/mL (4.56 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.56 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.5 mg/mL (4.56 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.56 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明

• [1]. Golding SE, et al. Improved ATM kinase inhibitor KU-60019 radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvival signaling, and inhibits migration and invasion. Mol Cancer Ther. 2009 Oct;8(10):2894-902.

  [2]. McCabe N, et al. Mechanistic Rationale to Target PTEN-Deficient Tumor Cells with Inhibitors of the DNA Damage Response Kinase ATM. Cancer Res. 2015 Jun 1;75(11):2159-65.

Cell growth is determined by AlamarBlue. U1242 cells are serially diluted, allowed to attach for 6 h and then exposed to KU-60019 at 3 μM. At days 1, 3 and 5 after seeding, AlamarBlue is added to the medium to the recommended final concentration. Plates are incubated for 1 h at 37°C and fluorescence determined on a FluoroCount plate reader (excitation 530 nm, emission 590 nm) and values taken as a measure of cell growth^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[2]
Cells (3×10⁷) are implanted into male Fox Chase Severe Combined Immunodeficiency (SCID) mice. Administration of Doxycycline is started when tumors reach 100 mm³ in volume and is performed every 48 hours up to removal of the animal from the experiment. Forty-eight hours after PTEN induction, animals are administered KU-60019 (100 mg/kg) for 5 consecutive days and measured until they reach a target 400 mm³ volume. Measurements of tumor volume and body weight took place every 3 days using calipers.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Golding SE, et al. Improved ATM kinase inhibitor KU-60019 radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvival signaling, and inhibits migration and invasion. Mol Cancer Ther. 2009 Oct;8(10):2894-902.

  [2]. McCabe N, et al. Mechanistic Rationale to Target PTEN-Deficient Tumor Cells with Inhibitors of the DNA Damage Response Kinase ATM. Cancer Res. 2015 Jun 1;75(11):2159-65.

KU-0063794 is a potent and specific mTOR inhibitor, inhibiting both the mTORC1 and mTORC2 complexes with IC₅₀s of 10 nM.

Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibits phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). Ku-0063794 induces a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppresses cell growth and induced a G1-cell-cycle arrest^[1]. Ku0063794 does not alter nuclear phospho-Mst1-Thr-120 levels in LNCaP cell nuclei, whereas Ku0063794 or CCI-779 increases phospho-Mst1-Thr-120 levels in C4-2 cell nuclei^[2]. The combination of GDC-0941 and KU0063794 inhibits the phosphorylation of 4EBP1 and S6 to a similar extent to that caused by single agent NVP-BEZ235 in HCT116, DLD1 and HT29 cell lines^[3].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

465.54

C₂₅H₃₁N₅O₄

938440-64-3

Room temperature in continental US; may vary elsewhere.

DMSO : 16.67 mg/mL (35.81 mM; Need ultrasonic)

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 1.67 mg/mL (3.59 mM); Clear solution

  此方案可获得 ≥ 1.67 mg/mL (3.59 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 16.7 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 1.67 mg/mL (3.59 mM); Clear solution

  此方案可获得 ≥ 1.67 mg/mL (3.59 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 16.7 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 3.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 1.67 mg/mL (3.59 mM); Clear solution

  此方案可获得 ≥ 1.67 mg/mL (3.59 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 16.7 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Garcia-Martinez et al. Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR). Biochem.J. (2009)421 29.

  [2]. Collak FK, et al. Threonine-120 phosphorylation regulated by phosphoinositide-3-kinase/Akt and mammalian target of rapamycin pathway signaling limits the antitumor activity of mammalian sterile 20-like kinase 1.J Biol Chem. 2012 Jul 6;287(28):23698-709. E

  [3]. Haagensen EJ, et al. The synergistic interaction of MEK and PI3K inhibitors is modulated by mTOR inhibition. Br J Cancer. 2012 Apr 10;106(8):1386-94.

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 0.5mol/LNaCl 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.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

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.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Garcia-Martinez et al. Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR). Biochem.J. (2009)421 29.

  [2]. Collak FK, et al. Threonine-120 phosphorylation regulated by phosphoinositide-3-kinase/Akt and mammalian target of rapamycin pathway signaling limits the antitumor activity of mammalian sterile 20-like kinase 1.J Biol Chem. 2012 Jul 6;287(28):23698-709. E

  [3]. Haagensen EJ, et al. The synergistic interaction of MEK and PI3K inhibitors is modulated by mTOR inhibition. Br J Cancer. 2012 Apr 10;106(8):1386-94.