Gel doc system

Stable HEK293T cell line expressing mutated FARSA protein that enables incorporation of azido-Phe into proteome of the cell was established using construct for piggybac transposase and PB-513B-1-GFP-MmPheT413G. Both plasmids were co-transfected into HEK 293T cells using Lipofectamine 3000 and incubated for 72 h before puromycin selection (10 μg/μl). The presence of the construct was observed as continuous expression of GFP.
Cells were transfected with pcDNA3.1 construct encoding S1m, RFP-S1m and 32×C4G2-S1m using Polyjet reagent (SignaGen Laboratories, Frederick, USA). 24 h later azido-Phe (JennaBioscience, Jena, Germany) was added to a final concentration of 125 μM. After 24 h incubation cell lysates were collected in lysis buffer (1% SDS in PBS with protease inhibitors), sonicated 3 × 10 s at amplitude of 80%. 25 μg of protein was added to the copper-catalyzed Click reaction kit (Jena, Germany). The reaction was done according to the manufacturer’s protocol. Alexa 555 alkyl (Invitrogen, Waltham, Massachusetts, USA) was used for staining and imaged on GelDoc System (Bio-Rad, Hercules, California, USA). Gels were stained with colloidal Coomassie dye G-250 (Gelcode Blue stain reagent, Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s protocol and imaged on GelDoc System (Bio-Rad, Hercules, California, USA).

P7 cerebella were dissected in PBS and lobules IX and X separated from lobules I–VIII. Tissue was lysed in 8 M urea, 1% CHAPS, 50 mM Tris (pH 7.9) followed by the removal of DNA by centrifugation. Proteins (10 μg per lane) were resolved on a Mini-PROTEAN precast gel (Bio-Rad, United Kingdom) and transferred to a nitrocellulose membrane (Bio-Rad, United Kingdom). After blocking with 0.5% nonfat milk powder in TBS with 0.5% Tween-20 (TBST), the membrane was incubated with primary antibodies (rabbit anti–DAB-1 [C-terminal], Sigma-Aldrich, D1569, 1:5,000; rabbit anti-GAPDH, Abcam, ab181602, 1:10,000) in 5% BSA, TBST overnight at 4°C. After washing and incubation with HRP-conjugated secondary antibody (Millipore) for 1 hour at room temperature, HRP conjugates were detected using Clarity Western ECL reagent (Bio-Rad). Gels were visualized on a Bio-Rad gel doc system and images analyzed with Image Lab (Bio-Rad) and prepared using Adobe Photoshop.