Fig. 6

c-IAP1 loss follows etoposide treatment and is inhibited by p21WAF1. (A) Kinetics of (c-IAP1) down-modulation by etoposide. K562 control transfectants were exposed to etoposide for 0 to 48 hr and expression of c-IAP1 and poly (ADP-ribose) polymerase (PARP) determined by Western blots analysis. (B) Prevention of c-IAP1 loss by p21WAF1. Representative results of three independent experiments are shown. Control transfectants (Neo) and p21WAF1-inducible transfectants (IWAF) were preincubated in p21WAF1 inducer (IPTG) as indicated, then etoposide (+, 100 µM) or carrier (−, 0.1% DMSO−) were added as shown. Cells were harvested after 48 hr and 50 µg of protein for each condition was immunoblotted with polyclonal anti-c-IAP1 (shown). Equivalent loading in each lane was evident upon Ponceau S staining. The immunogen used for the c-IAP1 antibody shown (Santa Cruz-1867: KTASQRLFPGPSYQNIKSIC) is not shared with (CIAP-2). Monoclonal anti-c-IAP1 gave the same results. (C) c-IAP1 message is not altered by etoposide. Northern blots of cells exposed for 48 hr to medium (−), etoposide (E), IPTG (I) or both (IE). The slight increase in signal in the two right lanes reflects loading differences. DMSO, dimethylsulfoxide; IPTG, p21WAF1 inducer.