Storm 860 gel and blot imaging system

Total RNA was extracted using Trizol reagent (Invitrogen). RNA (20 ug) was separated by 15% PAGE/8 M urea/ 1x TBE buffer. Gels were electro-blotted to a Hybond-N⁺ membrane (GE Healthcare) and UV cross-linked twice. Oligonucleotide probes (Supplementary Table 10) were labeled using [γ-³²P]-ATP (Perkin-Elmer) and T4 PNK (New England Biolabs). Labeled probes were purified with the Quick spin-oligo columns (Roche) before hybridization. Hybridizations were performed at 42°C overnight in UltraHyb-oligo solution (Ambion). After hybridization, membranes were washed twice in 2 × SSC/0.1% SDS and exposed to the Phosphor Screen System (GE Healthcare). The screen was scanned in a Storm 860 Gel and Blot Imaging System (GE Healthcare). As loading control, an oligonucleotide probe complementary to the U6 small nuclear RNA was used. Signal intensities were quantified using the ImageQuant 5.2 software (Molecular Dynamics). The U6 signal intensity in each blot was used for normalization and calculation of expression ratios. The same loading control was employed for membranes used more than once.

Detection of α6β1 integrin and laminin was performed in SVZ primary neurospheres and BEC collected in lysis buffer as previously described (Rosa et al., 2010 (link)). Total brain proteins were used as a positive control for α6 and β1 integrins. Activation of the Notch pathway was evaluated in SVZ cells obtained from the dissociation of primary neurospheres (Neurocult chemical dissociation kit, STEMCELL Technologies) and plated, as single cells, at a density of 900 000 cells per well of a 6-well plate coated with poly-D-lysine (20 μg/ml) alone or coated with either laminin-1 (25 μg/ml, reference: L2020), fibronectin (5 μg/ml) or vitronectin (5 μg/ml) (all from Sigma-Aldrich). Cells were grown for 72 h in SFM and harvested in lysis buffer. Western blot was performed as previously detailed (Rosa et al., 2010 (link)) using primary and secondary antibodies as listed in Supplementary Tables 2, 3, followed by visualization using ECF™ reagent on a Storm 860 Gel and Blot Imaging System (GE Healthcare) or using fluorescence-conjugated secondary antibodies on an Odyssey® Infrared Imaging System (Licor Biosciences). Band intensities were measured using ImageJ (NIH Image).

Equal amounts of proteins were resolved by SDS-PAGE in 8–10% polyacrylamide gels. Proteins were transferred to PVDF membranes Amersham Hybond (GE Healthcare Life Sciences) blocked at room temperature (RT) for 1 h in ×TBS with 5% milk + 0.1% Tween-20 (TBS-T). Membranes were incubated with primary antibodies at 4 °C overnight before washing in TBS-T and incubated with secondary antibodies (1:10,000) of horseradish peroxidase (HRP)-conjugated donkey (anti-mouse, anti-rabbit, Jackson ImmunoResearch) for 120 min at room temperature. Following three time washes in TBS-T, the membranes were incubated with ECL western blot substrate (GE Healthcare Life Sciences). Signal was visualized on a Storm 860 Gel and Blot Imaging System (GE Healthcare Life Sciences). Antibodies used in western blotting experiments were the following: anti-PSD-95 (6G6-1C9, 1:1000; Cell Signaling Technology), anti-PSD-93 (N18/275-284, 1:1000 NeuroMab), anti-CAMK2a (6G9, 1:1000, Sigma-Aldrich), GluA1 (AB1504, 1:1000, Millipore), GluA2 (MAB397, 1:1000, Millipore) and anti-GPRASP2 (ab129417, 1:100; Abcam or 12159-1-AP, 1:1000; Proteintech). When indicated, anti-β-tubulin (T7816, 1:20,000; Sigma-Aldrich) or anti-β-actin (A5441, 1:5000; Sigma-Aldrich) antibodies were used as loading controls. Uncropped western blots are shown in Supplementary Fig. 10.