Gradient 4 12 precasted gels

Cytosolic, membrane fractions and total protein extracts from cultured astrocytoma cell lines were obtained as previously described8 (link)54 (link). Equal amounts of proteins (30 μg) were resolved on SDS-PAGE using gradient (4–12%) precasted gels (Invitrogen), and transferred onto a nitrocellulose membrane. Nitrocellulose membranes were blotted overnight at 4 °C using the following primary Abs diluted in PBS/3% BSA: anti-Kir4.1 pAb (1:800, Alomone Labs), anti-Xpress mAb (1:2500, Life Technologies), anti-β-DG mAb (1:25, Novocastra Lab), anti-caveolin-1 pAb (1:1000, Santa Cruz Biotecnology), anti-Kir5.1 pAb (1:500, Abcam), anti-flotillin mAb (1:1000, BD Transduction Laboratories), anti-syntrophin mAb (1:200, MA-1-745, Affinity BioReagents, CO), anti-TRPV4 pAb (1:200, Alomone Labs), anti-connexin-43 mAb (1:250, BD Transduction Laboratories), anti-Kir2.1 pAb (1:250, Alomone Labs), anti-aquaporin-4 mAb (1:200, Santa Cruz Biotecnology), anti-actin mAb (1:2000, Santa Cruz Biotecnology). As secondary Abs we used peroxidase conjugated anti-mouse or anti-rabbit Abs (1:10000; Thermo Scientific, MO), for 1 hour at room temperature (RT). Immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Thermo Scientific), according to the manufacturer’s instructions and exposed on X-ray films.