Fig. 5.

Secreted Gal-3 is predominantly soluble and not packaged in EVs. (A) Soluble Gal-3 is secreted from MGAT1-deficient sHeLa. Wild type, positive control and negative clones for MGAT1-deficient cells were incubated in serum-free medium for 24 h. The cells were collected and lysed, whereas the medium was subjected to differential centrifugation at 300, 3000 and 100,000 g. A sample of the medium was collected after each centrifugation step. Gal-3 was assessed in the lysate, the entire 100,000 g EV pellet and medium (supernatant). Actin was used as a loading control and control for cell lysis. Exposure times are indicated for comparison. The 100,000 g EV pellets were also analysed by western blotting for levels of glycosylated and nonglycosylated CD63. (B) Soluble Gal-3 is secreted from SLC35A2-deficient sHeLa cells. Wild type, positive control and negative clones for SLC35A2-deficient cells were incubated in serum-free medium for 24 h. Samples were treated as described in A. (C) Secreted Gal-3 from MGAT1 and SLC35A2 mutant CHO Lec cells is soluble. Wild type (Pro5), MGAT1 (Lec1), MGAT1 rescue, SLC35A2 mutant (Lec8) and the double mutant (Lec3.2.8.1) were incubated in EX-CELL 325 PF CHO for 48 h, and the cells and medium collected. The cells, 100,000 g EV pellet and medium were processed as in A. Gal-3 and actin were analysed by western blotting and exposure times are indicated for comparison. CD63 was not analysed in this experiment as it does not cross-react with hamster CD63. Note that the same actin loading control blots are shown in the top panel of B and the bottom panel of Fig. 3A as the blots were stripped and reprobed with the indicated antibodies. Also, the top panel of A and the top panels of Fig. 3A and C, are the same blots.