CTLA4 is secreted via exosomes in response to v-ATPase inhibition. (A) Representative western blots of lysates and culture supernatants from mock- and Concanamycin A-(ConcA, 100 nM) treated A2058 cells (O/N: overnight). Cells were lysed and the media collected by serial centrifugation to concentrate extracellular vesicles (100k pellet, exosome fraction). (B) Quantification of CTLA4, ALIX, Syntenin, and CD63 recovered in the exosome fractions (100k) for data represented in A. Individual data points from two independent, color-coded experiments are shown. Error bars indicate range. (C) Predicted topology of CTLA4 in exosomal membranes. F8: C-terminal (C-ter) cytoplasmic domain directed CTLA4 antibody; TM, transmembrane domain; N-ter, N-terminal domain. (D) Representative western blots and Ponceau staining of samples from a Proteinase K protection assay of exosome-associated CTLA4. IB: Immunoblot. Source data are available for this figure: SourceData F2.
Figure 2.

CTLA4 is secreted via exosomes in response to v-ATPase inhibition. (A) Representative western blots of lysates and culture supernatants from mock- and Concanamycin A-(ConcA, 100 nM) treated A2058 cells (O/N: overnight). Cells were lysed and the media collected by serial centrifugation to concentrate extracellular vesicles (100k pellet, exosome fraction). (B) Quantification of CTLA4, ALIX, Syntenin, and CD63 recovered in the exosome fractions (100k) for data represented in A. Individual data points from two independent, color-coded experiments are shown. Error bars indicate range. (C) Predicted topology of CTLA4 in exosomal membranes. F8: C-terminal (C-ter) cytoplasmic domain directed CTLA4 antibody; TM, transmembrane domain; N-ter, N-terminal domain. (D) Representative western blots and Ponceau staining of samples from a Proteinase K protection assay of exosome-associated CTLA4. IB: Immunoblot. Source data are available for this figure: SourceData F2.

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