Fig. 1: Surface binding of GAPDH leads to aggregation of EVs.

a Cartoon representation of lactoferrin N cleavage from DC-LAMP by MMP-2 enzyme, and attachment of the cleaved lactoferrin on the EV surface via GAPDH protein. b Western blotting of purified HEK293T EVs and cell lysate showing expression of lactoferrin N-DC-LAMP and cleavage of lactoferrin from DC-LAMP (Dendritic cell-Lysosomal associated membrane protein). Arrangement of the different domains of the protein is shown in the schematic. The experiment was independently repeated two times. c UV-absorbance spectrum for EVs, EVs+GAPDH and EVs+BSA after passing through gel-filtration column. GAPDH and BSA protein alone were used as negative controls to rule out aggregation of the proteins. The experiment has been independently repeated three times. d Western blot showing exogenous binding of GAPDH to HEK293T EVs. Increasing concentrations of histidine-(His6) and Flag-tagged GAPDH were incubated with a fixed number of EVs. Endo GAPDH Endogenous GAPDH, Exo GAPDH exogenous GAPDH. Alix and CD81 are EV protein markers used as positive controls. Calnexin (bottom blot) was used to demonstrate the purity of EV samples. The experiment has been independently repeated two times. e GAPDH activity assay of EVs and EVs+Exo GAPDH. For EVs+Exo GAPDH activity, GAPDH protein was added to EVs in concentrated (10×) complete tissue culture media (Media + 10% FBS) followed by gel filtration to isolate EVs. Results are shown as mean ± s.d (n = 3 independent biological experiments). f NTA profile showing the size distribution of purified HEK293T EVs after incubation with either GAPDH or BSA proteins respectively. Inset is the scatter plot representing size (mean) of EVs. Data shown as mean ± s.d. (n = 3 independent biological experiments) Statistical differences were determined by unpaired two-sided Student’s t-test. ***P < 0.0001. g Electron microscopy images of EVs incubated with either BSA or GAPDH protein (scale bar indicates 200 nm). h Photographic images of tubes showing the formation of thread-like structures from MSCs and HeLa EVs after incubation with GAPDH for 2 h at 4 °C. Experiment of (g and h) were independently repeated two times. Source data are provided as a Source Data file.