Ecltm western blotting detect reagents

Cells cultured in the 3D-collagen gel were recovered following enzymatic dissociation with collagenase 1 (2 mg/mL) and whole cell lysates were prepared by incubation with radioimmunoprecipitation (RIPA) assay buffer (Santa Cruz) supplemented with a cocktail of protease inhibitors (Santa Cruz) and the total protein concentrations were determined using bicinchoninic acid (BCA) method (BioVision). Then 30 µg of proteins were subjected to SDS-polyacrylamide gel electrophoresis before being electroblotted onto a 0.2 μm nitrocellulose membrane (GE Healthcare). After blocking with 5% nonfat dry milk in TBST [25 mmol/L Tris (pH, 7.4), 137 mmol/L NaCl, 0.5% Tween20], membranes were incubated at 4 °C overnight with following primary antibodies: p75 (1:1000, Cell Signaling), NOCTH3 (1:1000, Abcam), HES1(1:1000, Cell Signaling). GAPDH (1;2000, Cell Signaling) was used as a loading control. After extensively washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz), and blot signals were developed with ECL^TM Western Blotting Detect Reagents (GE Health Care). All blots were derived from the same experiment and processed in parallel. Uncropped Western blotting images were provided in Supplementary Fig. 8 with the size markers labeled.

Cell lysates were prepared by incubation with radioimmunoprecipitation assay buffer (Santa Cruz) supplemented with a cocktail of protease inhibitors (Santa Cruz) and the total protein concentrations were determined using bicinchoninic acid method (BioVision). Then 30 µg of proteins were subjected to SDS-polyacrylamide gel electrophoresis before being electroblotted onto a 0.2 μm nitrocellulose membrane (GE Healthcare). After blocking with 5% nonfat dry milk in TBST [25 mmol/l Tris (pH, 7.4), 137 mmol/l NaCl, 0.5% Tween20], membranes were incubated at 4 °C overnight with following primary antibodies: LGR5 (Invitrogen, PA5-35304), ALDH1 (BD Biosciences, 611194), OCT4 (Abcam, ab18976), β-catenin (Cell Signaling, 8480S), ABC (Cell Signaling, 8814S), cyclin A (Sigma, C4710), cyclin B (Sigma, C8831), cyclin D1 (Cell Signaling, 2926) and cyclin E (Cell Signaling, 4129), ZEB1 (Santa Cruz, sc-25388), fibronectin (Sigma, F3648), and E-Cadherin (BD Biosciences, 562869). β-actin (Santa Cruz, sc-47778) was used as loading control. After extensively washing, membranes were incubated with HRP-conjugated secondary antibodies (Santa Cruz) and blot signals were developed with ECL^TM Western Blotting Detect Reagents (GE Health Care).

Planar-cultured GMSCs or astrocytes induced from GMSCs were harvested and whole-cell lysates were prepared by incubation with radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz) supplemented with a cocktail of protease inhibitors (Santa Cruz) and the total protein concentrations were determined using bicinchoninic acid (BCA) method (BioVision). Then 30 µg of proteins per well were subjected to SDS–polyacrylamide gel electrophoresis before being electroblotted onto a 0.2 μm nitrocellulose membrane (GE Healthcare). After blocking with 5% nonfat dry milk in TBST (25 mmol/L Tris, pH, 7.4, 137 mmol/L NaCl, 0.5% Tween20), membranes were incubated overnight at 4 °C with following primary antibodies: GFAP (1:1000, ab53554, Abcam), glutamine synthetase (1:1000, ab64613, Abcam), or GAPDH (1:2000, #5174, Cell Signaling) as loading control. After extensively washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz) and blot signals were developed with ECL^TM Western Blotting Detect Reagents (GE Health Care) and scanned using Amersham Imager 680.