Detection of OX40 and 4-1BB heterodimer by SDS-PAGE under a nonreduced condition. (A) HEK293 cells were transfected with OX40-HA and 4-1BB–FLAG as indicated on the top of figure. One percent NP-40 cell extracts were characterized by HA- or FLAG-specific Western blotting under reduced or nonreduced conditions. In subpanels i-ii, monomer of OX40 (∼48 kDa) is indicated by filled arrows. In subpanels ii and iv, presumed dimer of OX40 (∼96 kDa) and dimer of 4-1BB (∼60 kDa) are indicated by open arrows. Blots for presumed heterodimer (∼78 kDa) and the dimer of heterodimer (∼160 kDa) were observed in both HA-specific and FLAG-specific Western blots and indicated at right side in subpanels ii and iv. (B) The OX40-HA and 4-1BB–FLAG double-transfectant HEK cell extracts were immunoprecipitated with Abs specific for HA, FLAG, OX40, 4-1BB, or isotype control (indicated below panels). Immunoprecipitates were characterized by OX40, 4-1BB, HA-tag–or FLAG-tag–specific Western blotting under nonreduced conditions. Blots for presumed heterodimer (∼78 kDa) were observed in both OX40-HA–specific and 4-1BB-FLAG–specific Western blotting and indicated at the right side of each panel.