Background
MRT68921 is an inhibitor of ULK1 and ULK2 (IC50s = 2.9 and 1.1 nM, respectively).1 Through its effects on ULK1, MRT68921 blocks autophagy in cells, driving the accumulation of stalled early autophagosomal structures.1
1.Petherick, K.J., Conway, O.J.L., Mpamhanga, C., et al.Pharmacological inhibition of ULK1 kinase blocks mammalian target of rapamycin (mTOR)-dependent autophagyJ. Biol. Chem.290(18)11376-11383(2015)
Protocol
Kinase experiment: | Kinase assays are carried out in 50 mM Tris-HCl, pH 7.4, 10 mM magnesium acetate, 0.1 mM EGTA, and 0.1% β-mercaptoethanol, containing 30 μM cold ATP, and 0.5 μCi of [γ-32P]ATP for 5 min at 25 °C. Prior to ATP addition, reaction mixes are pre-warmed to 25 °C for 5 min. Reactions are stopped by the addition of sample buffer, followed by SDS-PAGE, transfer to nitrocellulose, and analysis by autoradiography and immunoblot[1]. |
Cell experiment: | MEFs and 293T cells are grown in DMEM, supplemented with 10% fetal bovine serum and penicillin/streptomycin, and cultured at 37°C, 5% CO2. For induction of autophagy, cells are typically grown to 75% confluency, ished twice, and incubated in Earle's balanced salt solution (EBSS) for 1 h (or complete medium as a control). MRT67307 (10 μM), MRT68921 (1 μM), AZD8055 (1 μM), or bafilomycin A1 (50 nM) is included[1]. |
参考文献: [1]. Petherick KJ, et al. Pharmacological inhibition of ULK1 kinase blocks mammalian target of rapamycin (mTOR)-dependent autophagy. J Biol Chem. 2015 May 1;290(18):11376-83. |
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