Anti β3 integrin

For immunoblot analysis of DITNC1 cells or primary astrocytes, cells were washed with ice-cold PBS and lysed with ice-cold lysis buffer (150 mM NaCl, 0.1% SDS, 0.25% sodium deoxycholate,1% Triton-X100, in 50 mM Tris-HCl pH, 7.0) supplemented with protease and phosphatase inhibitors (1 mM sodium orthovanadate, 2 μg/ml antipain, 1 μg/ml leupeptin, 10 μg/ml benzamidine and 1 mM PMSF). Protein extracts (30 μg) were mixed with Laemmli buffer, boiled for 5 min, electrophoretically separated on 10% SDS-PAGE and transferred to nitrocellulose membranes (Millipore). For Syndecan-4 detection, protein extracts were first digested with Heparitinase III (0.5 mU) for 3 h at 37°C and then mixed with Laemmli buffer. The membranes were blocked with TBS-T 5% fat-free milk and subsequently incubated with anti-Δ-heparan sulfate (1:2500; Seikagaku), anti-β₃ integrin (1:3000; Millipore), or anti-β-actin (1:3000; Sigma-Aldrich) antibodies for 1 h at room temperature, followed by the appropriate horseradish peroxidase-conjugated secondary antibodies. In all cases, peroxidase activity was revealed with the chemiluminescence kit (Pierce, Thermo Scientific). Immunoblot quantification was performed by measuring band intensity using ImageJ software and normalized to the loading control (β actin).

Protein extracts were prepared in a lysis buffer (150 mM NaCl, 0.1% SDS, 0.25% sodium deoxycholate, 1% Triton-X100, in 50 mM Tris-HCl pH 7.4) supplemented with a protease and phosphatase inhibitor cocktail (Biotool, Houston, TX, USA). Extracts were electrophoretically separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA), which were blocked with 5% w/v nonfat, dry milk in PBS containing 0.1% Tween-20, and subsequently incubated with anti-β3 Integrin (Millipore, Billerica, MA, USA) or anti β-actin (Sigma-Aldrich) primary antibodies. The membrane was then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Labs, Inc., West Grove, PA, USA) or goat anti-mouse IgG polyclonal antibody (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 1 h at room temperature. Bands were visualized with a chemiluminescence kit (Pierce, Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s instructions.

Cells were detached using trypsin/EDTA and incubated at 4 °C to avoid internalization of surface proteins. After blocking with BSA 5%, cells were immune-labeled with anti-β3 integrin (Millipore, Billerica, MA, USA) for 60 min. Cells were then washed and incubated with the anti-rabbit Alexa 488 secondary (Molecular Probes) for 30 min. Both incubations were at 4 °C. Cells were analyzed using a FACS Canto (BD Bioscience, San Jose, CA, USA) flow cytometer. Data were analyzed and plotted using FlowJo software (version v10.0.7, Stanford, CA, USA).