Roti fluoro

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.

GBM cells were lysed with a commercially available buffer containing Triton X-100 and protease/phosphatase inhibitors (both from Cell Signaling Technology, Frankfurt am Main, Germany). Cell debris was removed by centrifugation and the lysates were incubated with an SDS-Loading buffer containing 4% glycerin, 0.8% SDS, 1.6% beta-mercaptoethanol and 0.04% bromophenol blue (all from Carl Roth). Samples were separated by SDS-PAGE followed by transfer to Immobilon-P (Merck Millipore) or Roti^®-Fluoro (Carl Roth) PVDF membranes. The membranes were incubated with the following primary antibodies: anti-Catenin D1, anti-CD44, anti-CD99 (from Cell Signaling Technology), anti-MT1-MMP and anti-PLOD2 (from Proteintech) overnight at 4 °C. Secondary reactions were performed for 1h at room temperature using HRP-, AlexaFluor^®488- or AlexaFluor^®647-coupled antibodies (all from Cell Signaling Technology). All antibodies were diluted as recommended by the respective manufacturer using the SignalBoost™ Immunoreaction Enhancer Kit (Merck Millipore). Signal detection was performed on a ChemoStar imaging system (Intas Science Imaging, Göttingen, Germany).