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Puromycin metabolic incorporation assay
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hNPC cultures in one well of a six-well plate treated with DMSO or BBGD were pulsed with 1 µM puromycin (Sigma) for 1 h and then processed for western blots as described above using an anti-puromycin antibody (EQ0001, KeraFast - 1:1000). The puromycin incorporation, representing protein synthesis during the pulse phase, was determined by measuring total lane signal from 15–250 kDa. Signals were quantified using Empiria (LI-COR), normalized to total protein levels using Revert total protein stain (LI-COR), and presented as percent change relative to DMSO control.
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