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Fig. 3 | Molecular Neurodegeneration

Fig. 3

From: LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate

Fig. 3

LRRK2 phosphorylates NSF. a In vitro radioactive kinase assays of 3x-Flag LRRK2 wild-type, K1906M (kinase dead) and G2019S (hyperactive) and flag-NSF purified from HEK293T cells at 1:10 ratio. Radioactivity incorporated was revealed by autoradiography (upper panel) and total proteins loaded by flag immunoblotting (lower panels). LRRK2 inhibitor IN-1 was used at 1 μM concentration to confirm LRRK2 specific phosphorylation on NSF. b In vitro kinase assays as in (a) with the hyperactive GST-LRRK2970–2527 fragment. c Quantification of moles of 33P incorporated by NSF using a calibration curve with known concentration of 33P-ATP. d In vitro kinase assays as in (a) using NSF full-length or domains as substrates of LRRK2 GST-LRRK2970–2527 kinase activity at 1:10 ratio LRRK2:NSFs. Radioactivity incorporated was revealed by autoradiography (upper panel) and total proteins loaded by coomassie brilliant blue (CBB) staining for NSF (middle panel) or LRRK2 immunoblotting (lower panel). e In vitro radioactive kinase assays of 3xFlag-LRRK1 or 3xFlag-LRRK2 and Flag-NSF as substrate at 1:10 ratio. Left panel is an example of autoradiography and right panel represents the corresponding immunoblot of total loading

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Molecular Neurodegeneration

ISSN: 1750-1326