Complete protease and phosphatase inhibitor tablets

Hek293t cells were co-transfected using linear PEI (1 mg/ml) with plasmids encoding HA-Gtpbp2, Myc, Myc-Gsk3b, and/or Myc-Axin. Cells were incubated overnight and lysed in NP40 lysis buffer (10 mM Tris-Cl pH 8.0, 137 mM NaCl, 10 % Glycerol, 1 % NP-40, + complete protease and phosphatase inhibitor tablets; Roche). Cell lysates were incubated with mouse anti-Myc (9E10) antibody overnight, purified using anti-IgG magnetic beads (NEB) according to the manufacturers instructions. Immunoprecipitations and whole cell lysates (1:10 of IP volumes) were blotted with mouse anti-myc (1:100) and rabbit anti-HA antibodies (1:500). For western blotting Xenopus embryos or explants were lysed in NP40 lysis buffer and extracted with an equal volume of 1,1,2-trichlorotrifluoro-ethane (Sigma), and then separated using SDS-page. Detection of western blots was conducted with IR secondary antibodies (Alexa 680 and IRdye800) scanned on a Licor Odyssey Classic Imager. Levels of ectopic myc-Axin were normalized to levels of co-injected HA-mCherry.

MLi-2 was synthesized by Natalia Shpiro (University of Dundee) as described previously (Fell et al., 2015 (link)). HG-10-102-01 was from Calbiochem. Doxycycline, γ-S-GTP, HA-agarose and trypsin from Sigma and LysC from Wako. GluC, AspN and Chymotrypsin from Promega. GFP-agarose beads were from Chromotek. Complete protease and phosphatase inhibitor tablets were from Roche.

Cis-2,6-dimethyl-4-(6-(5-(1-methylcyclopropoxy)-1H-indazol-3-yl)pyrimidin-4-yl)morpholine (MLi-2) [13 (link), 37 (link)] was synthesised at the University of Dundee and used at a concentration of 200 nM for a duration of 30 min for LRRK2 kinase inhibition in the peripheral blood neutrophil experiments. Diisopropylfluorophosphate (DIFP) was purchased from Sigma (Cat# D0879), Microcystin-LR was from Enzo Life Sciences and sequencing grade modified trypsin from Promega (Cat# V511A). Complete protease and phosphatase inhibitor tablets were from Roche. All heavy and light stable isotope synthetic peptides described in supplementary Table 1 were synthesized by JPT peptide technologies (https://www.jpt.com/) in 1 nmol aliquots. All synthetic peptides were quantified by amino acid analysis and liquid chromatography–mass-spectrometry (LC–MS) analysis by JPT and confirmed to be of purity of > 95%. The peptides were delivered in a lyophilized form and were resuspended in solvent containing 0.1% (v/v) formic acid in 3% (v/v) acetonitrile to give a final concentration of 10 pmol/ml. Aliquots of this were further diluted in a series of tenfold dilution to a lowest concentration of 10 fmol/ml and stocks of each dilution aliquoted and stored at − 80 °C.