Supersignal west femto plus chemiluminescent substrate

A Western blot was performed to assess the concentration of pro-angiogenic proteins in the PLT-releasate of 4 HDs and 6 GBMs. Specifically, the protein amount was measured by Pierce Detergent Compatible Bradford Assay Kit (Thermo Fisher Scientific). An equal amount of protein (50 µg) was separated in Bolt 10% Bis-Tris Plus Gel (Thermo Fisher Scientific) in a Mini Gel Tank (Thermo Fisher Scientific) and transferred onto nitrocellulose iBlot 2 Transfer Stacks using the iBlot 2 Dry Blotting System (Thermo Fisher Scientific). After transfer, the membrane was blocked in Tris-buffered saline/Tween 20 + 5% milk solution and incubated separately with anti-GAPDH (SantaCruz Biotechnology), anti-VEGF (Abcam), anti-VEGFR2 (Abcam), anti-VWF (Thermo Fisher Scientific), or anti-S1P (Sonepcizumab, Creative Biolabs) overnight at 4 °C. After incubation with HRP-labeled secondary antibody (Invitrogen, Carlsbad, California, USA), the protein bands were scanned using SuperSignal West Femto PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) and detected by iBright5000 ChemiDoc XRSþ (Thermo Fisher Scientific). Densitometric analyses were performed using ImageJ [21 (link)].