Anti itgb1

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.