Anti puromycin clone 12d10

To analyze total protein synthesis, a SUnSET assay was performed. Therefore, primary cortical neurons were treated with 10 μg/ml puromycin (Merck) for 10 min at 37°C and 5% CO₂.
For immunoblotting experiments, neurons were harvested in 2× Laemmli buffer 7 days after transduction (DIV7 + 7). Samples were incubated at 95°C for 5 min and run on a 12% SDS–PAGE or 10%–20% tricine gels (Novex). The following primary antibodies were used for immunoblotting: anti-calnexin (ADI-SPA-860F; Enzo Life Technologies), anti-puromycin (clone 12D10, MABE343; Merck Millipore), anti-RPS6 (sc-74459; Santa Cruz Biotechnology), anti-RPL19 (sc-100830; Santa Cruz Biotechnology), and anti-RPL36A (sc-100831; Santa Cruz Biotechnology). For quantitative analysis, ImageJ was used and statistical analysis was done using the GraphPad Prism (version 7.01) software.

Frozen cell pellets were lysed in 50 mM Tris–HCl, pH 7.5, 10% glycerol, 5 mM MgCl₂, 1 mM EDTA, and 0.5% CHAPS. Protein content of lysates was determined by Pierce 660 nm protein assay reagent (ThermoFisher Scientific) and used to normalize gel loading. Lysates were run on NuPAGE gels (Invitrogen) using either MOPS or MES (Fig. 4e and Fig. S3d) running buffer before being transferred to a PDVF membrane, which was then blocked with Odyssey Blocking Buffer (LiCor), or 5% fat-free milk. Following antibodies were used: anti-K48-linkage Specific Polyubiquitin (D9D5, Cell Signaling, #8081), anti-actin (Abcam ACTN05(C4), or Cell Signaling 8H10D10 Cat #3700), anti-Bcl-X_L (Cell Signaling #2764), anti-Bcl-2 (Dako, # M0877), and anti-Mcl-1 (BD Pharmigen, # 559027), anti-Puromycin (clone 12D10, EMD Millipore cat #MABE343), HRP-conjugated anti-mouse IgG (Cell Signaling #7074), HRP-conjugated anti-rabbit IgG (Cell Signaling #7076), IRDye800CW labelled anti-mouse-IgG (Licor, #926-68070), Alexa680-labeled anti-rabbit-IgG (Invitrogen, #A21076). Bands were revealed using SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) followed by imaging using a CCD camera (GelDoc (Bio-Rad) or Azure c600 instruments) or on Odyssey scanner (LiCOR) using auto-exposure settings.

The protein synthesis rate was determined as previously described using either ³⁵S radioactive metabolic labelling or a Puromycin incorporation assay^{2 (link)}. Briefly, for ³⁵S methionine/cysteine labelling, cells were starved in methionine-free media supplemented with 10% dialysed FBS for 45 min. After starvation, the cells were incubated for 40 min in starvation medium supplemented with 30 μCi ml⁻¹ protein labelling mix (EasyTag protein labeling mix, Perkin Elmer). For the puromycin incorporation assay, the cells were treated for 30 min in culture medium supplemented with 1 μM puromycin. After treatment, the cells were harvested, lysed in RIPA buffer and the labelled proteins were run on an SDS–PAGE gel and blotted on a polyvinylidene fluoride membrane. The membranes were either exposed to autoradiography films (GE Healthcare) or incubated with anti-puromycin (clone 12D10, Merck Millipore).