TNF-α–induced apoptosis of 4-1BB/OX40-transfected cells. HEK293 cells were transfected with OX40-HA or 4-1BB–FLAG or both or control pcDNA3 as indicated on the left. Sixteen hours later cells were stimulated with TNF-α in the presence and absence of Abs against OX40 or 4-1BB. Fifteen hours later, cells were stained with FITC–annexin V to determine the proportion of cells undergoing apoptosis (indicated as percent in the panels of flow cytometry results). Alternatively, cell extracts were analyzed by SDS-PAGE for PARP-specific Western blotting that detects native PARP (115 kDa, shown as the top bands in panels on right) and bottom bands (89 kDa) indicated by arrows resulting from the caspase cleavages.