Fig. 6.

The secretion of intracellularly localized polySia-NCAM of human lung epithelial cell line A549 is induced by IL-1β and LPS. a Western-blot analyses of cell lysates of human A549 cells were performed using a mAb against polySia and NCAM (123C3) before and after endoN treatment. b, c Cultured A549 cells were fixed with methanol and stained with fluorescently labeled inactive endoN and b anti-NCAM mAb 123C3 or c polyclonal antibodies to visualize the trans-Golgi apparatus (anti-TGN38). For nuclear staining DAPI was used. d In addition, polySia was visualized in cells after stimulation with IL-1β (200 ng/ml) or LPS (100 ng/ml). Scale bars for c and d = 25 μm. e, f Polysialylated proteins were purified from supernatants of differently treated A549 cells and visualized by Western blotting using an anti-polySia mAb and after depolysialylation with a mAb against NCAM (123C3). g In an analogous cell lysates of IL-1β-treated cells were analyzed. h Western blots against polySia and NCAM (after depolysialylation) were performed using supernatants of untreated A549 cells as well LPS-stimulated cells in the absence or presence of TAPI-2. Apparent molecular masses of standard proteins are indicated in kDa