Standard methodologies were used. Protein extracts were separated by SDS–PAGE electrophoresis, and transferred to a PVDF membrane (GE Healthcare). Membranes were incubated using specific antibodies including mouse anti-puromycin PMY-2A4 (1:500 DSHB), mouse anti-GAPDH (1:2000, Thermo Fisher), mouse anti-Vinculin (1:2000, Sigma) and rabbit anti-FMRP (1:1000) (73 (link)).
Secondary HRP-conjugated anti-rabbit, anti-mouse antibodies (1:10 000) were purchased from Promega or Chemicon. Proteins were revealed using an enhanced chemiluminescence kit (Bio-Rad) and the imaging system LAS-4000 mini (GE). Quantification was performed using the IQ ImageQuant TL software (GE Healthcare). Coomassie staining of the membranes, GAPDH and Vinculin signals were used as normalizers.
Secondary HRP-conjugated anti-rabbit, anti-mouse antibodies (1:10 000) were purchased from Promega or Chemicon. Proteins were revealed using an enhanced chemiluminescence kit (Bio-Rad) and the imaging system LAS-4000 mini (GE). Quantification was performed using the IQ ImageQuant TL software (GE Healthcare). Coomassie staining of the membranes, GAPDH and Vinculin signals were used as normalizers.