Fusion fx7

The Western blot analysis was performed as described before [24 (link)]. The cells were collected in 75 µL of sample buffer. Ten micrograms of the sample were loaded on the electrophoresis gel. Anti-GAPDH (37 kDa) antibody (Cell Signaling, Cambridge, UK, 14C10, 1:1000) was used as housekeeping protein and PCNA (Santa Cruz, Dallas, TX, USA, 1:1000, PC10, 36 kDa) for the assessment of proliferation. The imaging and evaluation of blots was performed using the Fusion FX7 (PeqLab, Erlangen, Germany).

Protein concentration was determined by a Bio-Rad protein assay kit (Bio-Rad #500-0116) according to the manufacturer’s protocol. 10 μg of protein were fractioned by SDS-PAGE and transferred to a nitrocellulose blotting membrane (GE Healthcare, Germany). The membrane was washed three times with 1x Tris buffered saline with Tween20 (TBST) and incubated with 5% non-fat dry milk in TBST for 60 min. After three more washing steps with TBST, the membrane was incubated with rabbit anti-nestin (1:500, Abcam, Germany), rabbit anti-β-actin (1:2,000, EMD Millipore, Germany), rat anti-GFAP (1:1000, Invitrogen; Carlsbad, CA, USA), rabbit anti-p-AKT (1:2,000, EMD Millipore)and rabbit anti-Akt (1:2,000, EMD Millipore) at 4°C overnight. Membranes were washed three times for 10 min and incubated with a 1:5,000 dilution of anti-rat or anti-rabbit horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. Blots were washed with TBST three times and developed with the ECL system (GE Healthcare; Germany) according to the manufacturer’s protocols. Photos were taken with Fusion FX7 (Peqlab) and analyzed with ImageJ. Samples were blinded to experimenters and analysts.

Western blotting was performed as described previously (Aslam et al. 2010 (link)). Proteins were detected by FUSION‐FX7 (PeqLab) system and images were analysed using Quantity One software (Bio‐Rad).