Heterocomplex formation between OX40 and 4-1BB in gene-transfectant cells and T-cell blasts. (A) OX40 and 4-1BB coimmunoprecipitate from cell extracts derived from HEK293 transfectants. HEK293 cells were transfected with OX40-HA (full-length HA-tagged OX40) and 4-1BB-FLAG (full-length FLAG-tagged 4-1BB) as indicated on the top of the figure. One percent NP-40 cell extracts were immunoprecipitated with anti-FLAG (for 4-1BB immunoprecipitation) or anti-HA (for OX40 immunoprecipitation), then analyzed by HA-specific or FLAG-specific Western blotting as indicated. Lanes e-f show total cell lysates of OX40-HA–transfected HEK293 or nontransfected HEK293. Positions of OX40-HA and 4-1BB–FLAG are indicated by arrows on the right. (B) OX40 and 4-1BB coprecipitate by OX40L-GST or 4-1BB–GST from cell extracts of gene-transfectant cells. HEK293 cells were transfected with OX40-HA and 4-1BB–FLAG as indicated on the top of the figure. One percent NP-40 cell extracts were subjected to OX40-GST–or 4-1BB-GST–specific precipitation then analyzed by HA-specific Western blotting (WB). Lane a, precipitated by GST beads; lane b, precipitated by 4-1BBL–GST; lane c, precipitated by OX40L-GST; lane d, precipitated by 4-1BBL–GST; lane e, precipitated by OX40L-GST; and lane f, precipitated by GST. Position of OX40-HA is indicated by the arrow on the right.