Astrocyte Protein Isolation and Western Blot Analysis

Protein isolation from astrocytes was performed using M-PER buffer supplemented with protease and phosphatase inhibitors according to manufacturer’s protocol (Thermo Scientific, Rockford, IL, USA). Protein concentrations were measured using BCA protein assay (Thermo Scientific). Western blot was performed as described previously [32 (link)]. In short, equal amounts of protein (25-100 μg) were separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Bio-Rad Laboratories, Berkeley, CA, USA). After blocking in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, AKUSA), membranes were incubated with appropriate primary antibodies (for details, see Additional file 1: Table S1) overnight in Odyssey blocking buffer at 4°C. Primary antibodies were detected by incubation with appropriate IRDye secondary antibodies (LI-COR Biosciences) for 1 hour at RT in Odyssey blocking buffer and quantified using the Odyssey infrared imaging system (LI-COR Biosciences).