Expression of Bcl-xLin WEHI-231 immature B cells. (A) WEHI-231 B cells (107 cells/lane) were cultured with medium, anti-IgM (5 μg/mL), or anti-CD40 (10 μg/mL), either alone or in combination, for up to 48 hours. Cell lysates were prepared and Western blot analysis of Bcl-2, Bcl-xL, A1, or Mcl-1 expression was performed as described in “Materials and methods.” Experimental conditions were as follows: lane 1, medium 0 hours (C0), lane 2, anti-Ig 8 hours; lane 3, anti-CD40 8 hours; lane 4, anti-Ig plus anti-CD40 8 hours; lane 5, anti-Ig 24 hours; lane 6, anti-CD40 24 hours; lane 7, anti-Ig plus anti-CD40 24 hours; lane 8, anti-Ig 48 hours; lane 9, anti-CD40 48 hours; lane 10, anti-Ig plus anti-CD40 48 hours; and lane 11, medium 48 hours (C48). (B) Wild-type WEHI-231 cells were stimulated with anti-CD40 (10 μg/mL) for up to 48 hours, cell lysates were prepared, and Western blot analysis of Bcl-xL expression was performed as described in “Materials and methods.” Expression of Bcl-xL in anti-CD40–treated wild-type WEHI-231 cells was compared with that of unstimulated WEHI-231 cells transfected with the empty vector (Neo) or overexpressing Bcl-xL (Bcl-xL). Recombinant p42 Erk2 was also loaded as an additional standard. Data are representative of at least 3 independent experiments.
