Copyright information:Taken from "Novel link between E2F1 and Smac/DIABLO: proapoptotic Smac/DIABLO is transcriptionally upregulated by E2F1"Nucleic Acids Research 2006;34(7):2046-2055.Published online 14 Apr 2006PMCID:PMC1440883.© The Author 2006. Published by Oxford University Press. All rights reserved () Subcellular localization of ERE2F1. H1299 cells stably expressing ER-E2F1 fusion protein were examined by immunofluorescence staining using an E2F1 specific polyclonal antibody (H-137; Santa Cruz) in the absence (upper panels) or in the presence (lower panels) of 1 µM 4-OHT. The nuclei were stained with Hoechst 33342. () H1299 cells stably expressing ER-E2F1 were incubated in medium containing 0.1% serum for 48 h and then treated with 1 µM 4-OHT for the indicated periods of time. Total RNA was prepared from each treated samples and the equal amounts of RNA were employed in all the RT–PCR experiments. Smac/Diablo mRNA level was compared on 2% agarose gel. Cyclin A and β-actin mRNA levels were used as positive and negative controls, respectively. () H1299 cells stably expressing ER-E2F1 were treated similarly as in (b), and treated cells were harvested and lysed in RIPA buffer. An equal amount of total proteins from each time point indicated was employed for western blotting. The Smac/DIABLO protein levels were detected using a Smac-specific polyclonal antibody. Noxa and β-Actin protein levels were served as positive and negative controls, respectively.