Complete protease inhibitor cocktail tables

Proteins were extracted by standard procedures as previously described [34 (link)] in the presence of Complete Protease Inhibitor Cocktail Tables (Roche Applied Science). Control cells are SK-N-MC electroporated with the pLV-U6^#H1^#-C9G plasmid. Proteins were wet-transferred with TransFi (Invitrogen; Life Technologies) to polyvinyl difluoride (PVDF) membranes (Hybond-P, Amersham Biosciences) for 90 min. The protein-bound membranes were blocked with non-fat dry milk in Tris-buffered saline with Tween-20 at room-temperature and then incubated overnight at 4 °C with monoclonal mouse anti-human FOXP2 or MDFIC antibodies (1/1000 or 1/500; BD Pharmigen) or with rabbit anti-human GAPDH antibody (1/500; AbCam). After several PBS-T washes the membranes were incubated for 1 h at room temperature with secondary antibodies horseradish peroxidase (HRP)-conjugated with goat anti-mouse (1/1000) and goat anti-Rabbit (1/500; Dako, Barcelona, Spain) diluted in PBS-T. After several PBS-T washes the membranes were developed with enhanced chemiluminiscence (ECL) (GE Healthcare). The ECL signals were visualized on X-ray films. The FOXP2 and MDFIC proteins were normalized with the GAPDH internal control in the same lane.