Mouse anti lamp2

Automated immunostaining was carried out using Ventana BenchMarkXT platform (Ventana). The following antibodies were used; anti-vimentin and anti-cytokeratin (Ventana), rabbit anti-LC3A and rabbit anti-LC3B. Immunofluorescence was performed as described previously^{40 (link)}. For immunofluorescence, the following primary antibodies were used at the indicated concentrations: rabbit anti-LC3A (1:50) (Cat # 4599, Cell Signaling Technology), rabbit anti-LC3B (1:50) (Cat # 3868, Cell Signaling Technology), rabbit anti-vimentin (1:50) (Cat # 5741, Cell Signaling Technology), mouse anti-vimentin (1:100) (Cat # ab8978, Abcam), mouse anti-LAMP2 (1:50) (Cat # sc-18822, Santa Cruz Biotechnology), and mouse anti-LC3B (1:50) (Cat # sc-271625, Santa Cruz Biotechnology). Bound antibodies were visualized using Alexa Fluor 555 or Alexa Fluor 488 secondary antibodies (1:500) (Cell Signaling Technology). Cells were then counterstained using 4, 6-diamidinophenylindole (DAPI). Images were acquired using LSM 710 confocal scanning laser microscope (Carl Zeiss).

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).