Sodium azide

Cylin D1–APEX N2A cells were used to capture the proximity labeling reaction or collect purified cylin D1–APEX EVs. For the APEX reaction in receipt mESCs, cylin D1–APEX EVs were incubated with mESCs in N2B27 medium for 2 d at a concentration of ∼5 × 10⁹ EVs/ml medium. APEX proximity labeling was conducted as described previously (Hung et al., 2016 (link)). Biotin-phenol (500 µM) was preincubated with cells for 30 min at 37°C. Immediately before use, 1 mM (0.003%) H₂O₂ (Thermo Fisher Scientific) was spiked into the medium for the 1-min labeling reaction at RT. The reaction was then quenched immediately by three thorough washes with RT quencher solution, containing 10 mM sodium ascorbate (Sigma-Aldrich), 5 mM Trolox (Sigma-Aldrich), and 10 mM sodium azide (Sigma-Aldrich) in Dulbecco’s PBS (Thermo Fisher Scientific). Cells were lysed in radioimmunoprecipitation assay (RIPA) medium (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, and 0.1% SDS, pH 7.4) supplemented with 10 mM sodium ascorbate, 1 mM sodium azide, 1 mM Trolox, 1 mM DTT, and protease inhibitors (Roche). The whole-cell lysate was combined with loading buffer, heated at 95°C for 10 min, and resolved by SDS-PAGE. Biotinylated proteins were evaluated by blotting with streptavidin-HRP (21130; Thermo Fisher Scientific).