Anti mouse antibodies

Brain homogenates (10 μg protein), fibroblast lysates (10 μg protein), and EVs proteins (15 μl of the lysate corresponding to 25% of the EVs lysate total volume), were separated by 4–20% tris-glycine gel electrophoresis (Criterion precast gel, Bio-Rad, Hercules, CA) and transferred onto PVDF membranes (Immobilon, EMD Millipore, Billerica, MA). Membranes were incubated with antibodies to CD63 (1:250, Cat# sc-15363, Santa Cruz Biotechnology, Dallas, TX), Alix (1:1000, Cat# ABC40, EMD Millipore), TSG101 (1:1000, Cat# 4A10, GeneTex, Irvine, CA), Flotillin-1 (1:1000, Cat# 610821, BD Biosciences, San Jose, CA), Flotillin-2 (1:1000, Cat# 610384, BD Biosciences), rab35 (1:1000, Cat# 9690, Cell Signaling Technology, Danvers, MA), and β-actin (1:2500, Cat# ab8226, Abcam, Cambridge, MA). The secondary antibodies used were HRP conjugated anti-rabbit, and anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). Protein bands were quantified using ImageJ software (NIH, Bethesda, MD). All data are shown as the trisomic to diploid ratio for Ts2 and 2N mice, or for DS patients and 2N control subjects.

The cells were lysed 72 h after transfection in RIPA buffer, as well as in protease and phosphatase inhibitors (Sigma Aldrich Gmbh, Steinheim, Germany). Bradford assay determined the total protein concentration. Total protein from the lysates (30 μg) was resolved by 12% SDS-PAGE and, after electrophoresis, gels were blotted onto nitrocellulose membranes. Nonspecific binding sites were blocked for 2 h with 0.1% bovine serum albumin or 5% nonfat dried milk in TBST (TRIS-buffered saline solution containing 1% Tween 20). All washes and antibody incubations were performed using TBST. The following primary antibodies were used: anti-Cyclin E1 (Sta Cruz sc-481) 1 : 500 in blocking buffer (0.1% bovine serum albumin in TBST), anti-SHP (Perseus Proteomics-PP-N7519-00) 1 : 1000 in blocking buffer (5% nonfat dried milk in TBST), and anti-actinin 1 : 1000 in TRIS-buffered saline containing 1% Tween 20. Proteins were visualized by ECL detection with secondary HRP-conjugated anti-rabbit (Amersham Hybond ECL, Freiburg, Germany) or anti-mouse antibodies (Jackson Immuno Research, Pennsylvania, USA). Immunoblot results were quantified by densitometer using the GeneSnap and GeneTools software (SynGene-Synoptic Ltd., Cambridge, UK). Ponceau staining of membranes was used to monitor protein transfer and loading.