Necrostatin-1 (Nec-1)

Necrostatin-1 (1-100 μM) inhibits the autophosphorylation of overexpressed and endogenous RIP1.It is found RIP1 is the primary cellular target responsible for the antinecroptosis activity of this compound. [1]

This chemical efficiently suppresses necroptotic cell death triggered by an array of stimuli in a variety of cell types. It, previously identified as small-molecule inhibitor of necroptosis, inhibits RIP kinase-induced necroptosis and inhibits TNF-α-induced necroptosis in jurkat cells with EC50 of 490 nM. [2]

Necrostatin-1 (Nec-1) is a specific small molecule inhibitor of receptor-interacting protein kinase 1 (RIPK1) that specifically inhibits phosphorylation of this compound.

• RIP kinase 분석

  RIP1의 인산화에는 RIP1의 키나아제 활성이 필요합니다. FLAG 태그가 붙은 야생형(WT) 또는 키나아제 비활성 점 돌연변이 RIP1(K45M)의 발현 구조체를 293T 세포에 형질주입하고, [γ-³²P]ATP 존재 하에 30℃에서 30분 동안 방법(Methods)에 기술된 대로 RIP1 키나아제 분석을 수행합니다. 샘플을 SDS-PAGE에 적용하고 RIP1 밴드를 방사선 자동사진법(autoradiography)으로 시각화합니다. 방사성 밴드의 상대적 강도를 정량화하여 본 실험과 다른 모든 방사선 자동사진에서 (비율로) 나타냅니다. 키나아제 반응과 병행하여, 키나아제 반응에 포함된 단백질 양이 동일함을 확인하기 위해 비드 샘플을 anti-RIP1 항체를 사용하여 웨스턴 블롯 분석을 수행합니다.

• 세포주

  Jurkat, BALB/c 3T3, SV40-transformed MEF, L929

• 농도

  0.01-100 μM

• 배양 시간

  --

• 방법

  Cells are seeded in 96-well plates (white plates for luminescent assays; black plates for fluorescent assays; clear plates for MTT assay) at the density of 5,000-10,000 cells per well for adherent cells or 20,000-50,000 cells per well for suspension cells in 100 μl of the appropriate phenol red-free media. After incubation, we determined cell viability using one of the following methods. For the ATP assay, we used luminescence-based commercial kits and analyzed luminescence using a Wallac Victor II plate reader. For Sytox assay, we incubated cells with 1 μM Sytox Green reagent for 30 min at 37℃, and then performed fluorescent reading. Subsequently, we added 5 μl of 20% Triton X-100 solution into each well to produce maximal lysis and incubated cells for 1 h at 37℃, then performed the second reading. We calculated the ratio of values before and after Triton treatment and normalized it to the relevant controls not subjected to cytotoxic stimuli, as indicated in figure legends. For the MTT assay, we used the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit. For PI exclusion assays, we added 2 μg/ml PI into the medium and immediately analyzed samples using FACSCalibur. For PI-annexin V assay we used the ApoAlert Annexin V-EGFP Apoptosis Kit. For DioC6 staining, we incubated cells with 40 nM DiOC₆ for 30 min at 37 ℃, washed once and analyzed in FACSCalibur. For ROS analysis, we incubated cells with 5 μM dihydroethidium for 30 min at 37 ℃, washed once and analyzed in FACSCalibur. EM analyses are performed at the Harvard Medical School EM facility. We acquired bright-field images of the cells using an Axiovert 200 microscope.

• 동물 모델

  Male C57BL/6 mice

• 용량

  0.0468 mg/Kg

• 투여

  i.a.