Monoclonal puromycin antibody clone 12d10

Protein synthesis was measured using the SUrface SEnsing of Translation (SUnSET) method [39 (link)] which measures the incorporation of puromycin into nascent peptide chains. At 24 h after transfection, N2a cells were incubated in puromycin treatment media (1 μg/mL puromycin in DMEM (10% FBS, 1% P/S)). After a 30-min incubation at 37°C, puromycin treatment media was removed and cells were incubated with DMEM (10% FBS, 1% P/S) for 15 min. Cells were then washed with PBS two times and harvested for cell lysis. 20 μg of protein was separated on a 10% SDS-PAGE gel. Proteins were transferred to PVDF membrane using a Trans-Blot Turbo Transfer System (25 V, 1.3 A constant for 20 min; BioRad) and subsequently incubated in TBST buffer containing 5% BSA. Membranes were then incubated overnight with a monoclonal puromycin antibody (clone 12D10, EMD Millipore, Temecula, CA, United States, diluted 1:5000 in TBST and 5% BSA w/v), then washed three times with TBST and 1% BSA for 10 min each. Membranes were incubated for 2 h in TBST containing 3% skim milk plus a secondary mouse antibody (Sigma-Aldrich, 1:5000) then washed three times with TBST for 15 min each. Membranes were visualized on BioRad ChemiDoc imaging system. The total lane density was analysed using Image J [40 (link)].