FIG. 6.

Inducible expression of CREBR287L reduces CBE binding and viability of Ba/F3 cells. (A) Two Ba/F3 derivatives (clones 4 and 8) stably overexpressing CREBR287L under a Tet-off inducible system were established. The expression level of HA-CREBR287L (indicated by an arrowhead) in clones 4 and 8 in medium with (+) or without (−) tetracycline (Tet) was analyzed by immunoblotting with anti-HA and anti-CREB antibodies. C3, a Ba/F3 derivative expressing pTRE vector alone, is included as a negative control. (B) Decrease of CBE-binding activity in cells overexpressing CREBR287L. Nuclear extracts were prepared from each cell clone under conditions (with or without tetracycline) as indicated, and EMSA was then performed as described for Fig. 1 with a CBE (top) or GATA-1 (as an internal control; bottom) probe. The slow- and fast-migrating complexes are indicated as S and F, respectively. The asterisk indicates the position of the GATA-1 complex. (C) Suppression of cell viability by CREBR287L in stable lines. Cells cultured in the presence (+) or absence (−) of tetracycline for 72 h were washed and seeded in medium without IL-3. Sixteen hours after IL-3 depletion numbers of viable cell in each culture group were determined and are presented as percentages of the number of cells initially seeded. The data presented are averages from three independent experiments done in duplicate. ∗, 0.001 < P < 0.01 compared to lane 3; ∗∗, 0.001 < P < 0.01 compared to lane 5.