LRRK1 Immunoprecipitation kinase assay

Transfect HEK293 cells at around 60-70% confluency. For a 10cm dish, add 10µg DNA (GFP-tagged LRRK1 or GFP-empty vector) and 30µL of 1mg/mL PEI solution to 1mL of Opti-MEM^TM Reduced Serum Medium and vortex for 20/30 seconds.

Incubate at Room temperature for 0h 20m 0s to allow the DNA/PEI complexes to form.

Add the transfection mix to the culture medium in each dish and incubate cells at 37°C after transfection.

Lyse cells 20-24 hours after transfection.

Quickly rinse cells in the tissue culture dish by carefully pouring Room temperature culture media without Foetal bovine serum (FBS) into the dish.

Pour off media from the culture dish and completely aspirate any residual media. Immediately add freshly prepared ice-cold lysis buffer, ensuring that the entire surface is covered by lysis buffer.

Immediately transfer the cell dishes to ice.

Scrape the cells on the dish using a cell lifter (Sigma-Aldrich CLS3008, or equivalent) to ensure all cells are detached from the dish.

Using a pipette, transfer cell lysate to an Eppendorf tube On ice.

Leave samples On ice for 0h 20m 0s to allow for efficient lysis.

Clarify lysates by centrifugation at 20800x g,4°C.

Transfer the supernatants into new Eppendorf tubes and discard the pellet. Keep the tubes On ice.

Determine the protein concentration of cell lysates by Bradford assay according to the manufacturer’s instructions, performing measurements in triplicate.

Add 20mL of aGFP16-aGFP2-His6 NHS-activated Sepharose beads (washed 3 times in PBS and resuspended in PBS to make a 1:1 slurry) to 1mg of cell extract.

Incubate at 4°C for 3h 0m 0s, under mild agitation.

Collect the resin by centrifugation at 2500x g,4°C. Discard supernatant.

Resuspend resin in 500µL of lysis buffer supplemented with 300millimolar (mM) NaCl.

Repeat steps 16 and 17 twice.

Collect the resin by centrifugation at 2500x g,4°C. Discard supernatant.

Resuspend resin in 500µL of 50millimolar (mM) HEPES 7.5.

Repeat step 16 and 20.

Collect the resin by centrifugation at 2500x g,4°C. Discard supernatant.

Resuspend the resin in 50millimolar (mM) HEPES 7.5 (1:1 ratio).

Aliquot the resin into Eppendorf tubes kept On ice (one Eppendorf tube per reaction, 10µL resin each).

Prepare a “master mix” containing 50millimolar (mM) HEPES 7.5, 50millimolar (mM) KCl, 10millimolar (mM) MgCl2, 1millimolar (mM) ATP, and recombinant Rab protein.

Start the kinase reaction by adding the master mix to the immunoprecipitated kinase and transferring the Eppendorf tubes to the thermo mixer set at 30°C, 1000rpm,0h 0m 0s.

Stop the kinase reaction by adding 4X LDS loading buffer to the reaction mix to a final concentration of 2X.

Incubate the mixture at 70°C on a heat block for 0h 10m 0s to elute LRRK1 from the resin.

Collect the eluent by centrifugation through a 0.22‐μm‐pore‐size Spinex column.

Supplement the samples with 2‐Mercaptoethanol to 1% (v/v).

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
­AntibodyTarget	Company	Cat. number	Host species	Dilution
Rab7A (Total)	Sigma	R8779	Mouse	1 ug/ml
alpha-tubulin	Cell Signaling Technology	3873	Mouse	1:5,000
pS72 Rab7A	Abcam Inc.	MJF-38, Clone 1	Rabbit	1 ug/ml
LRRK1 (total) (C-terminus)	MRC-PPU Reagents and Services, University of Dundee	S405C	Sheep	1 ug/ml