Triazol

Neurons were grown and treated with various agents (proteasome inhibitors and other inhibitors) in Neurobasal A (Invitrogen). After treatment, the medium was changed to Neurobasal A lacking Methionine and supplemented with 4 mM AHA + / - treatments for 10 min. Neurons were washed 3x with PBS and lysed in PBS with 1% (w/v) Triton X100, 0.4% (w/v) SDS, protease inhibitors w/o EDTA (Calbiochem, 1:750) and benzonase (Sigma, 1:1000), heated at 95°C and centrifuged. BONCAT was performed as described in Dieterich et al. (2006) (link). In brief, 30 µg of total protein was subjected to a click reaction with 300 μM Triazol (Sigma, ref 678937), 50 μM biotin-alkyne tag (Thermo, ref B10185) and 83 μg/mL CuBr at 4°C o.n. in the dark. Biotinylated proteins were detected by Immunoblot. For puromycylation, puromycin was added to the culture media + / - treatments to a final concentration of 1.5 ng/µl for 10 min, then the neurons were washed 2x with PBS and lysed. For phosphorylation studies a phosphatase inhibitor cocktail (Invitrogen) was added to the lysis buffer. Tissue from HRI KO or wild-type mice was homogenized in lysis buffer (1% (w/v) Triton X100, 1% (w/v) SDS, protease inhibitors (1:750) and benzonase (1:500), heated to 95°C and centrifuged. SDS-PAGE was performed with 3 µg of lysate and proteins were subsequently blotted onto PVDF membranes.

Triazol was purchased from Sigma Chemical Co. (St. Louis, MO). The Girard
reagent GTr was synthetized dissolving Triazol (7.5 x 10^-3 moles)
in ethanol (20 mL) and mixed with ethyl chloroacetate (Aldrich) (9.4 x
10^-3 moles). The reaction mixture was stirred in reflux at
90°C for 72 hours and then was settled on ice and mixed with hydrazine
hydrate (2.9 x 10^-2 moles) under agitation for 20 minutes. The
solvent was evaporated in an ice bath, the residue was recrystallized in
methanol, and solid purified by column chromatography on alumina.

Larvae were incubated with 10 mM ANL for 24 hr. After incubation, the media was replaced with fresh E3. Larvae were sacrificed on ice-cold water (30 min). Heads were dissected using a scalpel and immediately snap frozen in tubes (1.5 ml, Eppendorf) that were pre-cooled using dry ice and stored at −80C until lysis. Tissue was homogenized and lysed using a pestle in lysis buffer (1% in Triton X100, 0.4% (w/v) SDS in PBS pH 7.8, 1:1000 EDTA free protease inhibitors (Calbiochem) and benzonase (Sigma, 1:1,000), and denatured at 75°C, 13,000 rpm for 10 min. Lysates were then cleared by centrifugation. BONCAT was performed as previously described (Dieterich et al., 2007 (link)). In brief, 60 μg proteins were dissolved in 120 μL PBS pH 7.8 supplemented with 0.01% SDS, 0.1% Triton, 300 μM Triazol (Sigma, 678937), 50 μM biotin-alkyne tag (Thermo, B10185) and 83 μg/mL CuBr (prepared by dilution of fresh 10 mg/mL solution in DMSO) at 4°C overnight in the dark. Biotinylated proteins were then separated by gel electrophoresis and immunoblotted with 1:1000 chicken anti-GFP (Aves), 1:1000 rabbit anti-biotin (Cell signaling) and Donkey anti-chicken IR680, goat anti-rabbit IR800 (IB, 1:10,000, Licor) antibodies.