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1 Biochemical Journal: this is an Accepted Manuscript, not the final Version of Record. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date version is available at Please cite using the DOI /BCJ BIOCHEMICAL JOURNAL ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Cite as Biochemical Journal (2016) DOI: /BCJ Copyright 2016 The Author(s). Use of open access articles is permitted based on the terms of the specific Creative Commons Licence under which the article is published. Archiving of non-open access articles is permitted in accordance with the Archiving Policy of Portland Press ( Use of open access articles is permitted based on the terms of the specific Creative Commons Licence under which the article is published. Archiving of non-open access articles is permitted in accordance with the Archiving Policy of Portland Press (

2 Phos-tag analysis of phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors

B FLAG-LRRK2 R1441G HA- WT T73A WT T73A MLi-2 + + + + + +

29 B FLAG-LRRK2 R1441G HA- WT T73A WT T73A MLi Coomassie staining Phos-tag gel (kda) 25 HA 37 total pt73 total LRRK2

30 A wild-type MEFs B 0.25 inhibitor: Phos-tag gel (kda) GSK HG MLi-2 LRRK2 activity [pmol/min/mg] IP Ab: UDD3 UDD3 UDD3 Pre-Immune IgG LRRK2: WT WT D2017A D2017A MLi-2: + IP: LRRK2 IB: LRRK2 IB: LRRK2 (Lysate) C MEFs: WT DMSO D2017A knock-in GSK A WT knock-in D2017A D MEFs: WT DMSO LRRK2 knockout GSK A WT LRRK2 knockout Phos-tag gel (kda) Phos-tag gel (kda) E MLi-2 MLi (nm) Phos-tag gel (kda) wild-type MEFs LRRK2[A2016T] knock-in MEFs

31 SK A (M) B HG (M) Phos-tag gel (kda) ild-type MEFs 37 wild-type MEFs GSK A D HG (M) (M) 2019S] knock-in MEFs Phos-tag gel (kda) LRRK2[G2019S] knock-in MEFs MLi (nm) 2019S] knock-in MEFs

2578215A 80 160 (min) B time wild-type MEFs

(min) Phos-tag gel (kda) 25 20 37 MLi-2 80

Phos-tag gel (kda) 25 20 G2019S MEFs + 1 M

32 A (min) B time wild-type MEFs + 3 M HG (min) Phos-tag gel (kda) MLi (min) D LRRK2 IP cell lysate time Phos-tag gel (kda) G2019S MEFs + 1 M GSK A (min) LRRK2 ps

33 A -in C MLi-2 mouse B-cells from wild-type + Phos-tag gel (kda) wild-type LRRK2[A2016T] knock-in (mg/kg)

A MEFs: WT DMSO GSK2578215A R1441G knock-in

37 MEFs: WT DMSO GSK2578215A G2019S knock-in

34 A MEFs: WT DMSO GSK A R1441G knock-in WT R1441G knock-in Phos-tag gel (kda) B 37 MEFs: WT DMSO GSK A G2019S knock-in WT knock-in G2019S Phos-tag gel (kda) 37

35 WT R1441G knock-in WT R1441G knock-in icle MLi-2 vehicle MLi-2

36 C DMSO GSK A IP: LRRK2 IB: LRRK2 ps910 IB: (Lysate) IB: (Lysate) IB: (Lysate) Phos-tag gel MEFs: (kda) WT S910A S935A knock-in WT S910A S935A knock-in 37 WT S910A S935A S910A S935A knockout knockout autoradiograph Coomassie IP: LRRK2 IB: IP: LRRK2 IB: LRRK2 Rab8A

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encouraged to use the Version of Record that, when published, will replace this version. The most /BCJ

Biochemical Journal: this is an Accepted Manuscript, not the final Version of Record. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date