Fig. 3.

GRASP KO reduces CS synthesis and secretion. A GRASP KO reduces CS synthesis analyzed by LC–MS. Shown is the total amount of CS in both cells and medium of indicated cell lines. B GRASP KO decreases CS synthesis analyzed by immunofluorescence microscopy. Indicated cells were permeabilized and stained for CS with a CS antibody (CS-56). Shown are microscopic images (left) and quantitation (right). C GRASP KO decreases CS synthesis shown by flow cytometry. Indicated cells were permeabilized, stained for CS with a CS antibody and analyzed by flow cytometry. D GRASP KO decreases CS secretion. The percentage of secreted CS (CS in conditioned media/CS in both cell lysate and conditioned media) was analyzed by LC–MS. E GRASP KO alters CS sulfation in the cell lysate. Shown is the percentage of each sulfated form of CS in cells. F GRASP KO alters CS sulfation in the conditioned media. Shown is the percentage of each sulfated form of CS in the conditioned media. Note that GRASP depletion increased 4-sulfation while decreased 6-sulfation in both the cell lysate (E) and conditioned media (F)