Figure 1.

Effects of apoptotic stimuli on Bim expression levels. (A) Flow cytometric analysis of Bim expression (rat mAb 10B12; solid black line) in thymocytes and spleen cells from wt and Bim^−/− (negative control; red line) mice that had been left untreated or treated for 22 h with ionomycin (2 µg/mL) or PMA (2 ng/mL). Staining with an isotype-matched control mAb was used as an additional control (black dotted line). (B) Bim protein expression was investigated by Western blot analysis (rat mAb 3C5) of lysates from unstimulated thymocytes (T0) and thymocytes cultured for 24 h in the presence of PMA (2 ng/mL), ionomycin (0.1 or 1 µg/mL), medium lacking FCS or in normal medium (control). (C) Expression of Bim (rabbit polyclonal Ab from Stressgen), Bcl-2, Mcl-1, pERK and HSP70 (loading control) was determined by Western blot analysis of lysates from untreated thymocytes or thymocytes that had been treated for 0.5, 1, 6 or 24 h with either PMA (P; 2 ng/mL) or ionomycin (I; 2 µg/mL). (D) Expression of Bim (rat mAb 3C5), Bcl-2, Mcl-1 and HSP70 (loading control) was determined by Western blotting of lysates from spleen cells that had been stimulated for 48 h with ConA (2 µg/mL) plus IL-2, followed by incubation with IL-2 for a further 48 h. These activated T cells were then deprived of IL-2 for the times indicated. The numbers underneath the anti-Bim blots in B, C and D indicate Bim protein counts/mm² relative to the loading control ((B) total Bim, (C and D) Bim_EL).