Immunoprecipitation of CLL B-cell lysates with MoAb to CD40L. (A) CLL B cells were cultured with media alone (lanes 1 and 2) or IL-4 at 10 ng/mL (lanes 3 and 4) and, after 36 hours, cell surface proteins were biotinylated and lysates were prepared. For each circumstance, lysates from 6 × 106 cells were subjected to immunoprecipitation using MoAb to CD40L (TRAP clone, lanes 1 and 3) or MoAb to IL-4 (lanes 2 and 4). As shown, the precipitated monoclonal Ig heavy chain (46 kD, designated “H”) and light chains (∼29 kD, designated “L1” or “L2” for the κ chains of the antibody to CD40L and to IL-4, respectively) are evident in addition to the CD40L-specific bands. The 39- and 32-kD bands, corresponding to the two transmembrane forms of CD40L, are indicated with thick arrows, and the soluble forms of CD40L, which include a doublet at 18 kD and an additional, single band at 15 kD, are indicated with narrow arrows. (B) CLL B cells were stimulated for 60 hours in culture with IL-2 and SAC, after which cell surface proteins were biotinylated and lysates prepared. In this experiment, 210 μg of protein were used in each immunoprecipitation, either with MoAb to CD40L (lane 1, TRAP clone) or to IL-4 (lane 2). (C) Lysates from cultured, superantigen-reactive, CD4+ T cells derived from a CLL patient were used for immunoprecipitation of CD40L after biotinylation of cell surface proteins. Lane 1 shows a 39-kD band after lysates from 1 × 106 T cells were used for immunoprecipitation. In lanes 2 (5 × 105 T cells) and 3 (2.5 × 105 T cells), the band is not evident.
