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2 protocols using iq imagequant tl software

1

Western Blot Analysis of Protein Extracts

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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.
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2

Western Blot Analysis of Protein Targets

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Standard methodologies were used. Protein extracts were separated by 10% or 8% SDS-PAGE and transferred to a PVDF membrane. Membranes were incubated using the following specific antibodies, including mouse anti-puromycin (1:500, DSHB), mouse anti-Vinculin (1:2000, Merck), mouse anti-GAPDH (1:2000, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-APP (1:2000, Merck), rabbit anti-ADAM10 (1:500, Abcam, Cambridge, UK), mouse anti-sAPPα (1:500, IBL America, Minneapolis, MN, USA), rabbit anti-OCT3/4 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-MAP2 (1:2000, Merck), mouse anti-Nestin (1:1000 Santa Cruz Biotechnology), mouse anti-SAP97 (1:1000, ENZO Life Sciences, Farmingdale, NY, USA) and rabbit anti-FMRP (1:1000, produced in house PZ1 [52 (link)]), HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (1:5000, Cell Signaling Technology, Danvers, MA, USA). Proteins were revealed using an enhanced chemiluminescence kit (Bio-Rad, Hercules, CA, USA) and the imaging system LAS-4000 mini (GE Healthcare, Chicago, IL, USA). Quantification was performed using the IQ ImageQuant TL software (GE Healthcare). Detection of GAPDH, Vinculin, and Coomassie staining were used as normalizers. For all SDS-PAGE PageRuler™ Plus Prestained Protein Ladder (10 to 250 kDa, Thermo Fisher Scientific) was used.
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