Cyclin D1 enriched in EVs during neurogenesis. (A) Immunoblot analysis of cyclin D1, 2, and 3 in PC12 cells induced by NGF for different times. D0, PC12 cells without NGF treatment. D1–D9, PC12 cells incubated with NGF for 1–9 d. (B) Immunoblot analysis of cyclin D1/2 in EVs purified from PC12 cells (D0) and EVs purified from NGF-induced PC12 cells for 3, 6, and 9 d (D3, D6, and D9). (C) Quantitative immunoblot analysis of protein levels described in B. The D0 signal was set as 1. Flot2 signal was used as a internal control. The values represent the mean ± SD, from three independent experiments (*, P < 0.05; **, P < 0.01). Error bars represent SD from independent samples. (D) Immunoblots of cyclin D1, Flot2, and Alix in EVs from undifferentiated ESCs (ES D0-EV) or 8-d (ES D8-EV) or 12-d (ES D12-EV) differentiated ESCs. (E) Immunoblots of cyclins, CDKs, Flot2, GM130, and actin in EVs and whole-cell lysates of PC12 cells or NGF-induced PC12 cells. (F) Immunoblot analysis of cyclin D1/2 and multiple EV markers of N6-EVs treated with different concentrations of proteinase K (PK), with or without 1% Triton X-100. (G) Immunoblots for cycinD1, Alix, Hsc70, Tsg101, and CD9 after immunoprecipitation of 5 × 10¹⁰ N6-EV with anti-CD9 antibody. IP, immunoprecipitates.