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Puromycin translation assay

TC Timothy S. Catlett
MO Massimo M. Onesto
AM Alec J. McCann
SR Sarah K. Rempel
JG Jennifer Glass
DF David N. Franz
TG Timothy M. Gómez
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The assay was performed as in Schmidt, Clavarino, Ceppi, and Pierre, 200932. Cultures were incubated in 10 µg/mL puromycin (ThermoFisher) for 10 min increments concurrent with or following treatment with Slit-2 (200 ng/mL), netrin-1 (100 ng/mL), or ephrin-A1 (200 ng–2 µg/mL) (R&D), and pharmacological agonists/antagonists as noted in the results, including rapamycin (Calbiochem) and anisomycin (MP Biomedicals). Puromycin-containing media was rinsed 2× with NB media before fixation. Growth cones were fixed and stained with anti-puromycin primary antibody (1:1000, PMY-2A4 was deposited to the DSHB by Yewdell, Jonathan (DSHB Hybridoma Product PMY-2A4)) and AF488 secondary (ThermoFisher) with AF-conjugated phalloidin (ThermoFisher) counterstain and analyzed as described above. All growth cones were normalized to TSC2+/+ puromycin-only control.

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