Figure S9. Extracellular vesicle (EV)-Cx43 channels are mostly closed.

(A) HEK293^Cx43− cells were transiently transfected with wild type Cx43 (Cx43^WT) or Cx43 mutated in the residues—S368A, S373A, or T154A—for 24 h. Cells were incubated in EV-depleted medium for 8 h. EVs were isolated, after which permeability was assessed by a modified transport-specific density shift technique. Cx43-containing EVs were layered onto a sucrose gradient (0.4–2.5 M) at pH 7 and ultracentrifuged. Sequential fractions were collected (from the top to the bottom of the tube), followed by density determination and washing with PBS by ultracentrifugation. Hsc70 was used as an EV marker (n = 3). (B) EVs isolated from cells overexpressing Cx43^WT, Cx43^S368A or Cx43^S373A were layered onto a sucrose gradient at pH 6 and ultracentrifuged. Hsc70 was used as an EV marker (n = 2).