Exogenous sphingomyelinase causes impaired intestinal epithelial barrier function

Figure 3 Preparations of detergent insensitive glycosphingolipid-enriched domains (DIGs) contain TJ-proteins. A: Lipid analysis of DIGs from non-stimulated Caco-2 cells by tandem mass spectrometry revealed the presence of high amounts of sphingomyelin, cholesterol and ceramide when compared to the total cell lysate; B: Gradient fractions were separated by 10% SDS-PAGE and analyzed by Western blot. Fraction No. 2 represents the raft-containing fraction at the 35%/5% interface (DIG); No. 9 represents the soluble fraction (cell lysate = CLy). The presence of high amounts of TJ-proteins occludin and claudin-4 and, to a lesser extent, E-cadherin show a clear association of TJ-proteins with DIGs. The simultaneous absence of other proteins like TLR4 and p38MAPK excludes significant contamination with non-raft fractions.