Biometra fastblot

Cells were harvested by scraping, washed in ice-cold PBS, and lysed in 150 μL/10 cm² lysis buffer (50 mM Tris-HCl pH 7.6, 250 mM NaCl, 0.1% Triton X-100, and 5 mM EDTA) supplemented with protease and phosphatase inhibitors (complete and PhosphoSTOP, Roche, Basel, Switzerland). Samples were sonified (Diagenode, Liège, Belgium) and cleared by centrifugation (15 min, 4 °C, 14000 × g). Relative protein content of supernatants was assessed using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein (typically 30 μg) were separated by SDS-PAGE and blotted (Biometra FastblotTM, Analytic Jena, Jena, Germany) onto nitrocellulose membrane (0.1 μm; GE Healthcare, Munich, Germany) by semi-dry blotting (1 mA/cm², 1 h). Primary antibody was applied in PBST (0.1% Tween-20) overnight at 4 °C. Membranes were washed thrice 10 min incubated with horseradish-coupled secondary antibody (1:2000) for 2 h at room temperature. After washing, ECL solution was applied (SuperSignal™ West Dura, Thermo Fisher Scientific) and specific bands were detected using a Stella gel documentation system (Raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany).

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.

Protein expression was analyzed by Western blot as described elsewhere [50 (link)]. Equal amounts of protein (typically 30–40 μg) were separated by SDS-PAGE and blotted (Biometra FastblotTM, Analytic Jena) onto nitrocellulose membrane (0.1 μm; GE Healthcare) by semi-dry blotting (1 mA/cm², 1 h). Quantitative densiometric analyses from Western blots were conducted with imageJ.