Nonfat milk powder

After quantification of the protein concentration using the Pierce BCA assay (Thermo Scientific, USA), protein lysates were separated on 12.5% SDS polyacrylamide gels and transferred to nitrocellulose membranes (0.2 µm, Amersham, USA) at 4 °C overnight. Equal loading of 7.5-µg protein per lane and transfer of proteins were confirmed by staining of membranes with Ponceau S solution (Sigma Aldrich). Nonspecific binding was blocked by incubation in 5% nonfat milk powder (Cell Signaling, USA) and 0.1% Tween-20 in tris-buffered saline (TBS) followed by incubation with the primary antibodies. The following primary antibodies were applied: rabbit anti-NeuN, goat anti-vascular cell adhesion molecule-1 (VCAM-1), rabbit anti-Iba-1, mouse anti-GFAP, and rabbit anti-glutaraldehyde-3-phosphate dehydrogenase (GAPDH) (Suppl. Table 7), each in blocking solution at 4 °C overnight. Membranes were incubated with appropriate peroxidase-conjugated secondary antibodies (all 1:5000, Dako, Denmark) in blocking solution at room temperature for 1 h followed by chemiluminescent detection with the enhanced chemiluminescence prime Western blotting detection reagent (Amersham, GE Healthcare Life Science, USA). For visualization and densitometric analysis, the ChemiDocXRS + imaging system and ImageLab software (Bio-Rad, Germany) were used.

Cells were cultured in 6-well plate (Corning), washed once with PBS (pH 7.4), and scraped using scraper (Fisherbrand). The suspension was lysed with 200 μl of lysing buffer supplemented with protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Thermo scientific) on ice for 60 min. Protein fractions were collected by centrifugation at 15,000 rpm at 4 °C for 10 min. Sample loading was normalized according to BCA (Bicinchoninic acid) relative protein quantification (Solarbio). Proteins separated following a NuPAGE 10% Bis-Tris Gel (Thermo), wet electrophoretic transfer was used to transfer the proteins to polyvinylidene difluoride (PVDF) membranes (0.45 µm; Millipore, Bedford, MA). The membranes were blocked with 5% non-fat milk powder (BD Bioscience) in Tris-buffered saline with 0.1% Tween (TBST) for 1 hour at room temperature and then incubated with ALDH1A1 antibody (rabbit mAb, Cell Signal Technology) or CXCR4 antibody (goat mAb, Abcam) overnight at 4 °C, followed by horse-radish-peroxidase conjugated goat anti-rabbit IgG or donkey anti-goat IgG (Cell Signal Technology) respectively for 1 hour at RT in 0.5% non-fat milk powder with TBST. Visualization was performed using Image Quant LAS 4000 with an Enhanced Chemiluminescence Kit (Thermo Pierce, Waltham, MA, USA).