ICSBP down-regulates bcl-2. For Western blot analysis protein lysates were harvested, separated on a 10% PAGE transferred to PVDF membranes, and immunoblotted using indicated antibodies. (A) Bcl-2 expression of clones derived from 32D/BA-v and 32D/BA-ICSBP (3A-D) by limited dilution (left) and 32D-v control and 32D-ICSBP cells (right). As a loading control the membrane was simultaneously also blotted with β-actin antibodies. (B) Transient expression of 32D cells with BCR/ABL (lanes 1-2) or 32D/BA cells with ICSBP (lanes 3-6). The percentage of reduction in bcl-2 expression was assessed by densitometric analysis as indicated. (C) Inverse correlation between the ICSBP and bcl-2 expression in 32D (left) as well as 32D-BA cells (right) with or without induction of ICSBP. ICSBP expression was turned off by addition of 1 μg/mL doxycycline for 48 hours. (D) Northern blotting: ICSBP causes a transcriptional down-regulation of bcl-2. Note the difference in the degree of bcl-2 regulation in BCR/ABL-positive and -negative cells; lane 1, 32D; lane 2, 32D/ICSBP; lane 3, 23D/BA; lane 4, 32D/BA/ICSBP-3E. (E) ICSBP does not affect the expression of other genes known to be involved in the regulation of apoptosis (lanes as described for panel D).