Dynasore (Dynamin Inhibitor I), a cell-permeable chemical, can reversibly and non-competitivity inhibit GTPase activity of dynamin 1/2 (IC50: 15 μM in a cell-free assay). It also inhibits the mitochondrial dynamin Drp1, with no effect against other small GTPase.

Dynamin1/2:15 μM, moderate potenc:~15 μM (vitro)

RIP1 kinase assay: Phosphorylation of RIP1 requires its kinase activity. Expression constructs of FLAGtagged wild-type (WT) or a kinase-inactive point mutant of RIP1 (K45M) are are transfected into 293T cells and RIP1 kinase assay is performed as described in the Methods in the presence of [γ-32P]ATP for 30 min at 30℃. Samples are subjected to SDS-PAGE and RIP1 band is visualized by autoradiography. Relative intensities of radioactive bands are quantified and are shown (ratio) in this and all other autoradiographs. In parallel to kinase reactions, a sample of beads is subjected to western blot analysis using anti-RIP1 antibody to ensure equal protein amounts in kinase reactions.

Mouse ventricular myocytes are isolated from male adult C6/Black mouse. Cardiomyocytes subjects to 2 hours of drug treatment followed by oxidative stress (30 μM Water2 for 35 min). For ATP supplement experiments, the cells are treated with 3 mM ATP for 30 min before exposure to Water2. Cardiomyocyte survival and viability are analyzed by trypan blue exclusion (TBE) assay[3].

Dynamin Inhibitor I

Ethanol: 1.6 mg/mL (4.96 mM), Sonication is recommended.

DMSO: 120 mg/mL (372.31 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 4 mg/mL (12.41 mM), Sonication is recommended.