Flotilin

Protein concentrations were determined using the BCA protein assay (Cat# PI23225, Thermo Fisher Scientific, Waltham, MA). Indicated samples were loaded on 4–20% Novex® gels (Cat# EC60285BOX, Thermo Fisher Scientific, Waltham, MA). Proteins were transferred for 1h at 120 V using TGS (25mM Tris pH=8.5, 192mM glycine, 0.1% (mass/vol) SDS), 20% (vol/vol) methanol as transfer buffer to polyvinylidene difluoride (PVDF) membranes 0.45μm (Cat# IPVH00010, Millipore, Billerica, MA), pre-activated in pure methanol. After transfer, the membranes were blocked at room temperature for 1 hour with TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1 % Tween 20), 5% (mass/vol) non-fat dry milk, then incubated separately in the primary antibodies VEGFR2 (Cat#PA5–16487, ThermoFisher), pY1175 VEGFR2 (Cat#PA5–105167, ThermoFisher), Flotilin (Cat#F1180, Sigma) or Neuropilin-1 (Cat#sc-5307, Santa Cruz biotechnology) in TBST, 5% (mass/vol) BSA, overnight at 4°C. Following incubation in horseradish peroxidase-conjugated secondary antibodies from Jackson immunoresearch, blots were revealed by enhanced luminescence (WBKLS0500, Millipore, Billerica, MA) before exposure to photographic film. Films were scanned, digitized, and quantified using Un-Scan-It gel version 7.1 scanning software by Silk Scientific Inc.