Anti phospho erk e 4

Cells and sEVs were lysed in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM NaF, 0.1 mM sodium orthovanadate) supplemented with protease inhibitor cocktail (cat. no., 25955; Nacalai Tesque). Lysates and immunoprecipitates were denatured in Laemmli buffer containing DTT and 2-mercaptoethanol, respectively. The denaturation was performed for 2 h at 37 °C for the lysates of EVs and for 5 min at 100 °C for the other samples. After cooling, the proteins were separated by SDS-PAGE, transferred onto PVDF membranes, blocked with 5% milk or 2% BSA for 1 h and probed with following primary antibodies: anti-Alix (12422-1-AP; Proteintech), anti-β-actin (clone AC-74; SIGMA), anti-CD9 (ab92726; Abcam), anti-ephrin-A1 (AER-031; Alomone Labs), anti-EphA2 (C-20; Santa Cruz), anti-Erk (#9102; CST), anti-FLAG (PM020; MBL), anti-p21 (C-19; Santa Cruz), anti-phospho-Erk (E-4; Santa Cruz), anti-phospho-p53 (Ser15) (#9284; CST), anti-phosphotyrosine (clone 4G10; Millipore), anti-PTP1B (clone FG6-1G; Millipore) and anti-Rab35 (11329-2-AP; Proteintech). All primary antibodies were diluted to 1 μg ml⁻¹. After washing, membranes were incubated with secondary antibodies (GE Healthcare) (1:1,000) for 1 h at room temperature (RT) and visualized with enhanced chemiluminescence reagents. Uncropped gel images are provided in Supplementary Figs 6–12.

HUVECs were lysed in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100). The lysate was incubated in SDS–PAGE loading buffer for 5 min at 95°C. The samples were separated on an SDS–PAGE gel and probed with primary antibodies (anti-Rab5B, 1:1000, Abcam; anti-cleaved Caspase 3, 1:1000, CST; anti-Caspase 3, 1:1000, CST; anti-phospho mitogen-activated protein kinase (anti-phospho-MEK) (S222), 1:1000, CST; anti-MEK, 1:1000, CST; anti-phospho extracellular signal-regulated kinase (anti-phospho-ERK) (E-4), 1:2000, Santa Cruz; anti-ERK1, 1:2000, Santa Cruz; anti-tubulin, 1:5000, Sigma) and visualized with enhanced chemiluminescent (ECL) HRP substrates (Amersham Biosciences, Litter Chalfont, U.K.). The intensity of bands was quantified with Image-Pro Plus 5.1 Image Analysis Software.