CTLA4 is secreted via exosomes from A2058 and HeLa S3 Flp-In CTLA4-HA cells. (A and B) Representative western blots of the lysates and cultured supernatants from mock- and Concanamycin A (ConcA)-treated A2058 and HeLa S3 Flp-In CTLA4-HA cells. Cells were lysed and media were subjected to trichloroacetic acid (TCA) precipitation. (C) Representative western blots of the lysates and cultured supernatants from mock- and ConcA-treated HeLa S3 Flp-In CTLA4-HA cells for indicated times. Cells were lysed and the media was collected by serial centrifugation to concentrate extracellular vesicles (100k pellet, exosome fraction). (D) Quantification of CTLA4, ALIX, Syntenin and CD63 secreted in the exosome fractions (100k) for data represented in C. Individual data points from three independent, color-coded experiments are shown. Error bars show SD. (E) Representative western blot of Cycloheximide (CHX; 100 µg/ml) chase experiment in HeLa S3 Flp-In CTLA4-HA and A2058 cells, following treatment with non-targeting (NT1) or ATG7 siRNA for 72 h. * indicates a non-specific band. (F) Quantification of blots represented in E. Error bars show SD from three independent experiments. Source data are available for this figure: SourceData FS2.