FIGURE 3.

Knockdown of Arrdc4 affects EV biogenesis. (a) Arrdc4 mRNA expression was examined by real time qPCR in HEK293Ts cells using β‐Actin as an internal control. +, comparing data with control; ⁺⁺ P < 0.01. (b, c) ANXA1 expression was examined by immunoblotting following Arrdc4 knockdown (using A4 siRNA) in HEK293Ts cells and re‐expressing Arrdc4^WT or Arrdc4^K270R as indicated. +, data compared with control siRNA; ⁺ P < 0.05, ⁺⁺ P < 0.01. (d) Nanoparticle analysis (NTA) showing EV_100‐200nm release normalised to equal number of cells. The Arrdc4 knockdown reduces EV production, whereas the re‐expression of Arrdc4^WT, but not Arrdc4^K270R significantly increases EV_100‐200nm release. +, data compared with untransfected control, whereas * indicates significance when compared with Arrdc4^WT. ^++/**P < 0.01, ns = not significant