Digital camera

Cells were lysed into 1× SDS loading buffer (50 mM Tris-HCl pH 6.8, 5% β-mercaptoethanol, 2% SDS, 0.01% bromophenol blue, 10% glycerol) followed by sonication (Bioruptor, 2 × 30 s at high setting). Proteins were solved on a 5–15% gradient Tris–glycine SDS–PAGE gel and semi-dry-transferred to nitrocellulose membranes. The following primary antibodies were used at the indicated dilutions: RAP1 (CST, 2399, 1:1,000); RASGRP3 (CST, 3334, 1:1,000), GAPDH (CST, 5174, 1:10,000); AKT (CST, 34685, 1:5,000); p-S473-AKT (CST, 4060, 1:2,000); ETS1 (CST, 14069, 1,000) and ETV2 (Abcam, ab181847, 1:1,000). All antibody information can be found in the Reporting Summary. Horseradish peroxidase (HRP)-conjugated secondary antibodies and the ECL prime western blotting system (GE Healthcare, RPN2232) were then used. Chemiluminescent signals were captured with a digital camera (Kindle Biosciences) and images of protein bands were taken for quantification using ImageJ.

A 10-cm plate of either HUVECs or ETV2-transduced HUVECs (flat 2D induction stage) was used for the active RAP1 assay (Cell Signaling, 8818S) according to the manufacturer’s guidelines for the kit. In brief, the cells were washed once with PBS and then starved for three hours in M199 medium with 0.5% BSA. The cells were then scraped in the lysis buffer supplied with the kit and resuspended at around 1 mg ml⁻¹. A fraction was saved as input and the rest of the cells were used for RAP1–GTP pull-down. Positive and negative controls, as well as a beads-only control, were performed according to the manufacturer’s guidelines. Proteins were solved on a 5–15% gradient Tris–glycine SDS–PAGE gel and semi-dry-transferred to nitrocellulose membranes. The membranes were then blocked in 5% milk in PBST and incubated in the provided RAP1 (1:1,000) antibody, GAPDH and/or ETV2 antibody for 48 h. After 48 h, the membranes were washed 3 times for 5 min and incubated in HRP-conjugated secondary antibody. Finally, after secondary washings, the membrane was blotted in ECL, chemiluminescent signals were captured with a digital camera (Kindle Biosciences) and images of protein bands were taken for densitometric quantification using ImageJ.