2OH-BNPP1 is an inhibitor of BUB1 kinase, a Ser/Thr kinase, used for the treatment of cancer.

BUB1 kinase^[1]

2OH-BNPP1 (0.1-50 μM) abrogates TGFβ signaling in a dose dependent manner. 2OH-BNPP1 dose-dependently impairs the TGFβ-induced phosphorylation of proteins that mediate canonical and non-canonical TGFβ signaling in multiple cancer cell lines and normal cell lines^[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.

2OH-BNPP1 (50 mg/kg) blocks TGFβ signaling in vivo, and significantly decreases abundance of phosphorylated SMAD2 in tumor-bearing mice^[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.

297.35

C₁₆H₁₉N₅O

833481-73-5

Room temperature in continental US; may vary elsewhere.

Powder	-20°C	3 years
	4°C	2 years
In solvent	-80°C	6 months
	-20°C	1 month

In Vitro: 

DMSO : ≥ 100 mg/mL (336.30 mM)

* “≥” means soluble, but saturation unknown.

*

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。

In Vivo:

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    90% corn oil

  Solubility: ≥ 2.5 mg/mL (8.41 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (8.41 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]. Nyati S, et al. The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-β signaling. Sci Signal. 2015 Jan 6;8(358):ra1.

In vitro kinase assays are performed using 200 ng of protein (BUB1-WT, or a kinase inactive mutant BUB1-KD), and 2 µg of substrate (TGFBRI-WT, TGFBRI-KD, SMAD3 or H2A) in 1X kinase buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl₂, 1% (v/v) Glycerol, 0.1% (v/v) Triton X 100, DTT, PMSF, Na₃VO₄ (1 mM each), 2 mM NaF and β-glycerol phosphate) in 20 µL volume containing 10 µCi ³²p-ATP and 300 µM cold ATP. Reactions are run at 30ºC for 0.5-1 hour and quenched using Laemmli buffer and resolved using a 4-12% Bis-Tris gel. Quantitative autoradiography is performed using a Typhoon FLA 9000 scanner.

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.

Cell line-derived xenografts are established by implanting 2.5×10⁶ A549 cells subcutaneously into each flank of 4-6 week old male SCID mice. When tumors reach a volume between 40 to 60 mm³, mice are injected with a single intraperetoneal dose of SB431542 (10 mg/kg body weight), a dose previously reported to inhibit TGFβ signaling in mouse models, or 2OH-BNPP1 (50 mg/kg), or vehicle (DMSO). Tumors are excised 4 hours after treatment and fixed in formalin. Paraffin-embedded sections are stained using an antibody for phosphorylated SMAD2, and micrographs are taken with an Olympus microscope fitted with an Olympus DP-70 high resolution digital camera. The proportion of nuclei that stains positive for phosphorylated SMAD2 are counted in three random fields per tumor per treatment condition [vehicle (n=4), SB431542 (n=2), and 2OH-BNPP1 (n=5)]. A two -ided Student’s t-test is performed to assess statistical significance. Slides are adjusted for brightness and contrast with Adobe Photoshop CS2, but the micrographs undergo no further manipulations.

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]. Nyati S, et al. The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-β signaling. Sci Signal. 2015 Jan 6;8(358):ra1.