Gadd45β suppresses apoptosis by Fas and is induced rapidly by CD40. (A) PI staining of representative Neo, HA-Gadd45β, and Bcl-xL BJAB clones treated with anti–APO-1 for 12 hours (top panels). Bottom panels show Western blots with extracts (50 μg) of BJAB clones and antibodies against HA, Bcl-xL, or β-actin, as indicated. (B) PI staining of representative HA-Gadd45β, Bcl-xL, DN-FADD, and Neo BJAB clones treated with anti–APO-1 for the times shown. (C) Rapid induction of Gadd45β by CD40. Western blots showing Gadd45β levels in the Fas-resistant clone HA-Gadd45β-109 (G109, lane 2; Neo-(N)124, lane 1) and in parental BJAB cells cocultured with CD40L fibroblasts (1:1 ratio) for the times shown (lanes 3-5). Lysates from parallel cultures of BJAB and CD40L cells alone indicated that, in lysates from mixed cultures, BJAB-derived proteins were 20% of total proteins. Accordingly, gels were loaded as follows: BJAB clones alone (lanes 1-2), 20 μg; cocultures (lanes 3-5), 100 μg (ie, 20 μg + 80 μg); CD40L fibroblasts alone (lane 6), 80 μg. Antibodies were as shown: anti-Gadd45β (5D2.2), anti–β-actin (controlling for loading of fibroblast proteins), or HRP-labeled anti–human IgG (controlling for loading of BJAB proteins). HA-Gadd45β, endogenous Gadd45β, and nonspecific (n.s.) bands are indicated. (D) c-FLIPL and Bcl-xL are not induced or are induced only modestly or slowly by CD40 stimulation. Extracts of CD40L-stimulated BJAB cells were the same used in (C). Loading was as in (C) (lanes 1-3, 100 μg; lane 4, 80 μg), and antibodies were as shown. L and S indicate c-FLIPL and c-FLIPS forms, respectively. (E) PI staining of Burkitt lymphoma (Raji and Ca46) and non-Burkitt lymphoma B cells transduced with MIGR1 or MIGR1-Gadd45β and treated with anti–APO-1 for the times indicated. Cells were FCM-sorted as in Figure 1C. (A, B, E) Values were extrapolated as in Figure 1 and represent the mean ± standard deviation of 3 independent experiments.
