Inducible expression of 4-1BB mRNA and protein in mouse mast cells. (A) BMMCs derived from 129/SvJ and C57BL/6 mice were left unstimulated (-) or incubated overnight with anti-DNP IgE 206. The IgE-sensitized cells were washed and incubated with PBS, 10 ng/mL DNP₂₃-HSA, 100 ng/mL SCF, or 1 μg/mL LPS for the indicated periods. RNAs were isolated from these cells and subjected to RT-PCR for the expression of 4-1BB, 4-1BBL, and GAPDH mRNAs. (B) 4-1BB expression was directly analyzed by immunoblotting of lysates of wild-type (wt) and 4-1B^-/- BMMCs with anti-4-1BB mAb clone 158321 (top). Alternatively, immune complexes precipitated with anti-4-1BB mAb clone 3H3 were analyzed by immunoblotting with anti-4-1BB polyclonal antibody (bottom). The broad (37-45 kDa) band of 4-1BB with the strongest signal around 40 kDa in both unsensitized and IgE-sensitized wt, but not 4-1B^-/-, cells is indicated by an arrow. (C) C57BL/6 BMMCs were sensitized with IgE and stimulated with antigen for the indicated periods as described in panel A. Flow cytometric analysis of surface expression of 4-1BB was performed. Continuous lines represent 4-1BB expression and broken lines isotype control. Results shown in panels A and B are representative of 3 independent experiments and those in panel C are representative of 5 similar experiments.