Figure 2.

Ectodomain shedding of transmembrane tumor necrosis factor–like cytokine 1A (TL1A) in human umbilical vein endothelial cells (HUVEC) and U-2OS cells. (A) HUVEC were transfected with TL1A cDNA or empty vector (Mock) by AMAXA electroporation, immunostained for TL1A, and examined by confocal fluorescence microscopy. Nuclei were stained with TO-PRO 3 (right). Expression of TL1A was verified by Western blot (left). (B) HUVEC were transfected with either empty vector (Mock) or a TL1A overexpression vector by AMAXA electroporation. HUVEC cell extracts and supernatant were examined by Western blot using antibodies specific for TL1A. (C) U-2OS cells were transiently transfected with TL1A cDNA and Mock using Lipofectamine 2000. After antibiotic selection for transfected cells, they were seeded at a density of 3 × 10⁵ per well in a six-well plate. Ten micromolars of TAPI-1 and the equivalent volume of DMSO as a control were added as indicated and incubated for 24 hours. Cell extracts and speed-vac concentrated supernatants were examined by Western blot using anti-TL1A antibody. (D) U-2OS cells were treated for 2 hours with 50 ng/mL phorbol-12-myristat-13-acetate (PMA) and 5 mM H₂O₂, as indicated. Micrographs of treated cells are shown. (E and F) U-2OS cells were transfected with TL1A cDNA and seeded as described previously. Cells were either untreated (NT) or treated with increasing concentrations of PMA (E) or H₂O₂ (F) in the presence or absence of TAPI-1 (20 μM), as indicated. After 2 hours, supernatants were harvested, concentrated by speed-vac evaporation, and subjected to Western blot analysis. (G) Supernatant of U-2OS cells overexpressing TL1A was concentrated via speed-vac evaporation and treated with the indicated enzymes to remove all N- and O-linked oligosaccharides. The enzymes were added sequentially, in a cumulative manner, so that in the end all five enzymes are added to the reaction (from left to right on the indicated Western blot). TL1A fragments were separated on a large sodium dodecyl sulfate–polyacrylamide gel electrophoresis for higher resolution and analyzed by Western blot using anti-TL1A antibody.