Assessment of PKC-dependent activation of LRRK1 in vitro

Clean a disposable glass culture tube by washing. Allow to air-dry.

Clean a disposable glass culture tube by washing with 100% methanol. (1/3)

Clean a disposable glass culture tube by washing with 100% methanol. (2/3)

Clean a disposable glass culture tube by washing with 100% methanol. (3/3)

Pipette 0.5µL of Diacylglycerol (stock concentration is 10mg/mL) and 5µL of Phosphatidylserine (stock concentration is 10mg/mL) into the cleaned and dried glass tube.

Vacuum dry lipids using a SpeedVac system for 0h 10m 0s. This should leave a visible, translucent lipid pellet.

Resuspend lipids in 50µL of 25millimolar (mM) HEPES 7.4, 50millimolar (mM) KCl. Vortex gently until pellet is no longer visible.

Prepare a primary “2X master mix” containing

A	B
HEPES pH 7.5	50 mM
KCl	100 mM
2‐Mercaptoethanol	0.2% (v/v)
MgCl2	20 mM
ATP	2 mM
CaCl2	2 mM
Phosphatidylserine	200 μg/ml
Diacylglycerol	20 μg/ml

For each reaction, add 10µL of the primary “2X master mix” to a clean Eppendorf tube.

Add 5µL of 200nanomolar (nM) LRRK1 wild type protein (final concentration is 50nanomolar (nM)) to each reaction and allow equilibration 30On ice for 0h 5m 0s.

Start the kinase reaction by adding 5µL of 400nanomolar (nM) PKC Alpha protein (final concentration is 100nanomolar (nM)).

After 0h 30m 0s, transfer the Eppendorf tubes from Step 8 30On ice.

Prepare a secondary “master mix” (=Master Mix B) containing

A	B
HEPES pH 7.5	25 mM
KCl	50 mM
MgCl2	10 mM
ATP	1 mM
Rab7A	1 μM

Start the second step of the kinase reaction by adding 10µL Master Mix B to the Eppendorf tubes from Step 5.

Transferring the Eppendorf tubes to the thermo mixer set at 30°C, 1,000 rpm. Incubate for 0h 45m 0s.

Stop the kinase reaction by adding 10µL of 4× LDS (supplemented with 5% (v/v) 2‐Mercaptoethanol) loading buffer to the reaction mix to a final concentration of 1×.

If using LRRK1 immunoprecipitated from cells, stop the kinase reaction by adding 30µL of 4× LDS loading buffer to the reaction mix to a final concentration of 2×, incubate the mixture at 70°C on a heat block for 0h 10m 0s to elute LRRK1 from the resin, and collect the eluent by centrifugation through a 0.22‐μm‐pore‐size Spinex column.

Incubate the samples for 0h 5m 0s at 70°C on a heat block before proceeding to quantitative immunoblotting analysis.

The reaction products can be analysed by quantitative immunoblotting analysis (as described in XXXX). Table 1 lists the primary antibodies that we recommend using, which include antibodies to detect Rab7A phosphorylation at Serine-72.

A	B	C	D	E
­Antibody Target	Company	Cat. number	Host species	Dilution
pS72 Rab7A	Abcam Inc.	MJF-38, Clone 1	Rabbit	1 mg/ml
Rab7A (Total)	Sigma	R8779	Mouse	1.430556
LRRK1 (total) (C-terminus)	MRC-PPU Reagents and Services, University of Dundee	S405C	Sheep	1 mg/ml
PKC Alpha	Abcam Inc.	ab11723	Mouse	1.430556

Figure 1: PKC alpha dose-dependent activation of recombinant LRRK1 in vitro. Recombinant LRRK1 wild type [27-2015] was incubated with increasing concentrations of PKC Alpha (1 to 300 nM) at 30°C for 0h 30m 0s with excess Mg-ATP. Reactions were subsequently incubated with 1micromolar (µM) recombinant Rab7A and subjected to a 0h 45m 0s kinase reaction at 30°C in the presence of excess Mg-ATP. Kinase reactions were subjected to immunoblot analysis with the indicated antibodies and the membranes were developed using the Odyssey CLx scan Western Blot imaging system.