Western blot analysis
Protein isolation
Protein extraction from cells was conducted using
radioimmunoprecipitation assay (RIPA) lysis buffer comprising of 50mM
tris-HCL, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulphate
(SDS), 0.5% sodium deoxycholate, 2mM ethylenediaminetetraacetic acid
(EDTA), half of a cOmplete™ protease inhibitor cocktail tablet
(Sigma-Aldrich, Cat. No. 11697498001) and one PhosSTOP™ phosphatase
inhibitor cocktail tablet (Roche, Cat. No. 04906837001). 50μL of buffer
was added to each cell pellet, samples were sonicated for 10 pulses at
10% followed by incubation on ice for 30 minutes before storage at
-80°C.
Mouse brains were dissected into ipsilateral and contralateral
hemispheres and the cortex and striatal regions were isolated from each
hemisphere. Samples were snap-frozen in liquid nitrogen and finely
ground using a mortar and pestle. The ground sample was then further
mechanically homogenised using a silicon microtube pestle at a tissue
weight to homogenisation buffer ratio of 1:4. The homogenisation buffer
comprised of 10% tris buffer (50mM tris hydrochloride (Chem-supply,
Cat. No. BIOTB0103) and 150mM sodium chloride (Chem-supply, Cat. No.
SA046)), 1% triton x-100 (Sigma-Aldrich, Cat. No. T8787), one cOmplete™
protease inhibitor cocktail tablet (Sigma-Aldrich, Cat. No. 11697498001)
and half of a PhosSTOP™ phosphatase inhibitor cocktail tablet (Roche,
Cat. No. 04906837001). The homogenised samples were placed on a
microtube roller for two hours at 4°C before centrifugation at 2000g for
10 minutes Supernatants were placed in fresh tubes and stored at -80°C
for protein analysis.
Western blot
Protein concentration was determined by Bradford assay. Isolated
proteins were incubated with 2X Novex™ tris-glycine SDS sample buffer
(Thermo, Cat. No. LC2676) containing 5% 2-mercaptoethanol
(Sigma-Aldrich, Cat. No. M3148) for 5 minutes at 90°C. 20-40μg of
protein was resolved on 10% acrylamide SDS-0 (PAGE) gels. Gels were
transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Cat.
No. 1704272) using a Trans-Blot® SD semi-dry transfer cell (Bio-Rad).
Membranes were then blocked in 5% w/v skim milk in tris-buffered saline
(TBS) with 0.05% Tween-20 (Sigma, Cat. No. P1379) (TBS-T) for one hour,
followed by an overnight incubation at 4°C with primary antibody
(Supplementary Table 1) diluted in 5% w/v skim milk. The membranes were
washed three times for 10 minutes each with TBS-T and incubated with
horseradish peroxidase (HRP) conjugated secondary antibody
(Supplementary Table 2) in 5% w/v skim milk for one hour at room
temperature. Membranes were washed again with TBS-T and signals detected
using an Amersham™ ECL™ prime western blotting detection kit (GE
Healthcare, Cat. No. GEHERPN2232) and imaged using a ChemiDoc™ imaging
XRS+ system (Bio-Rad). Band intensity was measured using Image Lab™
software (Bio-Rad, Version 6.0.1) and normalised to the β-actin loading
control. Values are presented as band intensity relative to β-actin.