Fig. 5: Linifanib prevents RIPK1-mediated cell death and is a RIPK1 kinase inhibitor.
From: Repositioning linifanib as a potent anti-necroptosis agent for sepsis
A FADD-deficient Jurkat cells were pretreated with linifanib (10 µM), Nec-1 (25 µM) or DMSO for 30 min followed by stimulation with hTNF-α (50 ng/mL) for the indicated time. The expression levels and activation status (phosphorylation) of the indicated necrosome members p-RIPK1S166 and p-MLKLS345 were analyzed by western blotting using the indicated specific antibodies. Data shown are representative of two independent experiments. B MEFs cells were pretreated with DMSO, linifanib (5 µM), Nec-1 (25 µM) plus Smac mimetic (200 nM) and z-VAD-fmk (20 µM) followed by stimulation with mTNF-α (25 ng/mL) for the indicated durations. C TNF-α-induced complex I immunoprecipitation (IP) of MEFs cells treated in the absence or presence of linifanib (0.31 µM) with Flag-mTNF-α (100 ng/ml) for the indicated time points, followed by anti-Flag beads and western blotting analysis using the indicated antibodies. Lysates pre-IP (input) were also analyzed by western blotting using the indicated antibodies. D In vitro ADP-Glo™ kinase assays using recombinant human RIPK1 protein (200 nM). Recombinant hRIPK1 was incubated with linifanib and Nec-1 at the indicated concentrations. Data represent mean value ±S.D. of two independent experiments.
