Anti rpl19

The mouse monoclonal antibody recognising the opsin tag (Adamus et al., 1991 (link)) and the rabbit antiserum against Sec61α (N-terminus) (Laird and High, 1997 (link)) were as described previously. Rabbit antisera against: SRP54, Sec61β and SRα were gifts from Bernhard Dobberstein (University of Heidelberg, Heidelberg, Germany); Sec61α (C-terminus) and Sec62 from Richard Zimmermann (University of Saarland, Homburg, Germany); and PPL from Sharon Tooze (Francis Crick Institute, London, UK). A rabbit antiserum recognising an internal peptide of the human 25-kDa subunit of the signal peptidase complex (SPCS2) was custom made by Eurogentec. Anti-calreticulin was purchased from Affinity Bioreagents (catalogue number, PA3-900). Anti-RPL19 was purchased from Santa Cruz Biotechnology (catalogue number sc-100830). Antibody dilutions are given in the relevant section.

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.