Chemostar imaging system

The total protein was extracted with a buffer that contained 150 mM NaCl, 1% Triton X-100, 0.5% sodiumdeoxycholate, 0.1% SDS and 50 mM Tris that was supplemented with a protease inhibitor cocktail (Roche, Penzberg, Germany). The total protein content was measured using a protein quantitation assay (Thermo Fisher Scientific) according to the manufacturer’s protocol. The total protein (20 µg) was loaded onto 10% denaturing SDS gel and transferred to 0.45 mm polyvinylidene fluoride membranes (GE Healthcare, Little Chalfont, UK) for immunoblotting. The membranes were blocked with 5% nonfat dry milk (Sigma-Aldrich) and probed with primary antibodies against VCAM-1, β-Actin, p-AKT, AKT, p-eNOS and eNOS, followed by horseradish peroxidase–labeled secondary antibodies. Proteins were detected using a chemiluminescence substrate (Bio-Rad Laboratories, Hercules, USA). The results were documented on a Chemo-star imaging system (INTAS, Göttingen, Germany). The signal intensity of the chemiluminescence was quantified using Quantity One software (Bio-Rad).

Proteins were extracted from six-well plates with 500 µl ice-cold RIPA buffer (10 mM Tris–HCl pH 8, 1 mM EDTA, 1% v/v Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) supplemented with protease inhibitors (1 mM Pefabloc, 1 ng/µl aprotinin/leupeptin, 10 mM BGP, 1 mM NEM). Protein samples were then sonicated three times for five cycles (30 s ON/30 s OFF) in a Bioruptor Pico (Diagenode, Belgium). Lysates were then mixed with Lämmli buffer (6 × , 375 mM Tris–HCl, 10% SDS, 30% glycerol, 0.02% bromophenol blue, 9.3% DTT) and cooked for 5 min at 95 °C. Same amounts of protein per sample were then separated using 10–12% SDS polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Immobilon, Millipore, USA). Membranes were blocked with 5% skimmed milk in TBS-T and incubated overnight at 4 °C with specific primary antibodies diluted in the same blocking solution. Next, membranes were washed with TBS-T, incubated 1 h with secondary antibodies (Dianova GmbH, Germany) diluted in blocking buffer, washed again with TBS-T and finally developed using HRP substrate (Cyanogen WESTAR) in a ChemoStar imaging system (INTAS science imaging, Germany). Antibody list in Additional file 1: Table S6.

GBM cells were subjected to lysis using a buffer containing Triton X-100 along with protease and phosphatase inhibitors (Cell Signaling Technology). The lysates were cleared by cellular debris through centrifugation and then mixed with a loading buffer containing 4% glycerin, 0.8% SDS, 1.6% beta-mercaptoethanol, and 0.04% bromophenol blue (all from Carl Roth). The proteins were electrophoretically separated by SDS-PAGE and then transferred to Immobilon-P (Merck Millipore, Darmstadt, Germany) or Roti^®-Fluoro (Carl Roth) PVDF membranes using a semidry blotting device (Biometra Fastblot^TM, Analytik Jena AG, Jena, Germany). The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-PGRMC1, anti-TCF 1/7, anti-beta-Actin (all from Cell Signaling Technology), or anti-ITGB1 (Proteintech, Manchester, UK). Subsequent secondary reactions were carried out for 1 h at room temperature with HRP-, AlexaFluor^®488-, or AlexaFluor^®647-coupled antibodies (all from Cell Signaling Technology). All antibodies were diluted in SignalBoost™ Immunoreaction Enhancer (Merck Millipore) according to the manufacturer’s recommendation. The ChemoStar imaging system (Intas Science Imaging, Göttingen, Germany) was used for the detection and acquisition of signals and the intensity of the bands was quantified with the ImageJ 1.48v software.