Figure 3

Localization of Nedd5 in HEp-2 cells and in EAhy926 cells. (a) Immunofluorescence analysis of Nedd5 localization in HEp-2 cells. The mouse polyclonal antiserum specific to Nedd5 C-ter was used to analyze the cellular distribution of Nedd5. (b) 10.5% SDS–PAGE and immunoblotting of EAhy926 cells. EAhy926 cells were centrifuged and the pellets were redissolved in the loading buffer under reducing conditions (100,000 cells/well). 10.5% SDS–PAGE stained by Coomassie blue (1); immunoblotting was performed with the mouse polyclonal antiserum specific to Nedd5 C-ter (2) and with the mouse preimmune serum (3). (c) Immunofluorescence analysis of Nedd5 localization in endothelial cells under physiological conditions and during apoptosis. EAhy926 cells were treated with tumor necrosis factor α (TNF-α) (20 μg/ml) plus cycloheximide (10 μg/ml) for 16 hours to induce apoptosis. The mouse polyclonal antiserum specific to Nedd5 C-ter was used to analyze the cellular distribution of Nedd5. (i) Untreated cells, fixed with 4% formaldehyde in PBS; (ii) TNF-α plus cycloheximide-treated cells, fixed with 4% formaldehyde in PBS; (iii) untreated cells, permeabilized with acetone:methanol 1:1 (vol:vol); (iv) TNF-α plus cycloheximide-treated cells, permeabilized with acetone : methanol 1:1 (vol:vol).