BMS-3-LIM-Kinase-(LIMK)-Inhibitor-MedChemExpress

BMS-3

LIMK inhibitor BMS-3 (0, 1, 10 and 50 μM). The increasing concentrations of BMS-3 result in a strong decrease on the

percentage of sperm that undergoes acrosomal exocytosis after stimulation with 20 μM of Progesterone[2].

PROTOCOL

Kinase Assay [1]The protein kinase domains of human LIMK1 and LIMK2 are expressed as glutathione S-transferase fusion proteins using the Bac-to-Bac system in Sf9 cells. Compounds 1 to 6 (e.g., BMS-3) are assayed for inhibition of LIMK1 and

LIMK2 protein kinase activity by radioactive phosphate incorporation into biotinylated full-length human destrin.

Reactions are done with a concentration series of compound in 25 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 5 mM

MnCl2, 1 μM total ATP, 83 μg/mL biotinylated destrin, 167 ng/mL glutathione S-transferase-LIMK1, or 835 ng/mL

glutathione S-transferase-LIMK2 in a total volume of 60 μL at room temperature for 30 min (LIMK1) or 60 min

(LIMK2). Reactions are terminated by addition of 140 μL of 20% TCA/100 mM sodium pyrophosphate, and the

precipitates are harvested onto GF/C unifilter plates. The radioactivity incorporated is determined using a TopCount

after addition of 35 μL Microscint scintillation fluid[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

REFERENCES

[1]. Ross-Macdonald P, et al. Identification of a nonkinase target mediating cytotoxicity of novel kinase inhibitors. Mol Cancer Ther. 2008 Nov;7(11):3490-8.

[2]. Romarowski A, et al. PKA-dependent phosphorylation of LIMK1 and Cofilin is essential for mouse sperm acrosomal exocytosis. Dev Biol. 2015 Sep 15;405(2):237-49.

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Caution: Product has not been fully validated for medical applications. For research use only.

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