Transturbo blot pvdf membranes

Whole cell lysates were used for Western blotting following conventional protocols for gel electrophoresis and trans-blotting, as described earlier in [12] . Equal amounts of 10 µL lysate containing 2 µg/µL protein were loaded on precast TGX stain-free gels (Bio-Rad, Munich, Germany). Transturbo blot PVDF membranes (Bio-Rad) were used for blotting. An overview of the used antibodies and their applied concentrations is given in Table 3. The analysis was performed using ImageQuant™ LAS 4000 (GE Healthcare UK Limited, Buckinghamshire, UK), and the densitometry was performed using ImageJ (NIH).

Whole cell lysates were used for Western blotting following routine protocols for gel electrophoresis and trans-blotting, as described earlier [31, 33] . Equal amounts of 10 µL lysate containing 2 µg/µL protein were loaded on precast TGX stain-free gels (Bio-Rad, Munich, Germany). Transturbo blot PVDF membranes (Bio-Rad) were used for blotting. Each Western blot contained 5 samples for each group: 4 h 1g, 4 h RPM-AD cells, 24 h 1g, 24 h RPM AD cells and 24 h RPM MCS.
The following primary antibodies were used at a dilution of 1:1000: fibronectin (F3648), laminin (L9393) and talin (T3287) (all Sigma-Aldrich) and vascular endothelial growth factor (VEGFA; ab46154, Abcam, Cambridge, United Kingdom). The corresponding secondary antibodies were used at a dilution of 1:3000: HRP-linked anti-mouse IgG (#7076) and anti-rabbit IgG (#7074, both Cell Signaling Technology, Massachusetts, USA). The analysis was performed in ChemiDoc XRS+ (Bio-Rad), and the densitometric quantification was performed using ImageLab (BioRad).