Recombinant human vegf a165

Primary HUVECs were cultured as previously described (Fearnley et al., 2014 (link); Howell et al., 2004 (link)), HDMECs and appropriate growth media were from PromoCell (Heidelberg, Germany). Purified primary and secondary antibodies were typically used at 1 µg/ml for microscopy and at 0.1 µg/ml for immunoblotting. These antibodies were goat anti-VEGFR2 (R&D Systems, Minneapolis, USA), rabbit anti-phospho-VEGFR2 (Y1175), rabbit anti-UBA1 (Cell Signaling Technologies, Danvers, USA), rabbit antibodies to native and phosphorylated PLCγ1 (Y783), rabbit anti-ERK1/2, mouse anti-phospho-ERK1/2 (T202, Y204), mouse anti-α-tubulin (Santa Cruz Biotechnology, USA), mouse anti-transferrin receptor (TfR), mouse FK2 anti-ubiquitin (Affiniti Research Products, Exeter, UK), mouse anti-EEA1 (BD Biosciences, California, USA), mouse anti-CD63 (Abcam, Cambridge, UK), mouse anti-LAMP2 (Santa Cruz, USA), HRP-conjugated secondary antibodies (Thermo Fisher, Loughborough, UK) and Alexa Fluor-conjugated secondary antibodies (ThermoFisher). Endothelial cell growth medium (PromoCell), non-targeting and UBA1 siRNA duplexes (GE Dharmacon, UK) and recombinant human VEGF-A₁₆₅ (Genentech Inc., San Francisco, USA) were obtained as stated. Chemicals were obtained from Sigma-Aldrich (Poole, UK) or Thermo Fisher (Loughborough, UK).

Antibodies: goat-anti-VEGFR1 (#AF321), goat-anti-VEGFR2 (#AF357), rabbit-anti-phospho-VEGFR2-Y1214 (#AF1766) (R&D Systems, Minneapolis, MN, USA), rabbit-anti-Akt (#9272S), rabbit-anti-phospho-Akt (S473; #4060B), rabbit-anti-ERK1/2 (#9102S), mouse-anti-phospho-ERK1/2 (T202/Y204; #9106S), rabbit-anti-neuropilin 1 (NRP1; #3725S), rabbit-anti-phospho-VEGFR2-Y951 (#4991S), rabbit-anti-phospho-VEGFR2-Y1059 (#3817S), rabbit-anti-phospho-VEGFR2-Y1175 (#2478S; Cell Signaling Technology, Danvers, MA, USA), rabbit-anti-phospho-VEGFR2-Y1054 (Clone D1W; #04-894; Merck Millipore, Watford, UK), mouse-anti-α-tubulin (Clone DM1A; #T6199; Sigma Aldrich, Poole, UK), mouse-anti-PECAM-1 (CD31; #sc-65260; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-ubiquitin (FK2; #14220; Caymen Chemical, MI, USA), mouse-anti-clathrin heavy chain antibody (X22; #ab2731; Abcam, Cambridge, UK), sheep-anti-TGN46 (#AHP500GT; AbD Serotec, Oxford, UK). Endothelial cell growth medium (ECGM) was from PromoCell (Heidelberg, Germany). Recombinant human VEGF-A₁₆₅ was from Genentech Inc. (San Francisco, CA, USA), both VEGF-A₁₂₁ and VEGF-A₁₄₅ was from Promocell.