HCA7 cells were grown on glass chamber slides and treated with SC236 (5 mM) or vehicle control for 24 h. Slides were air-dried, fixed (methanol) and permeabilised (cold acetone). Slides were then blocked with 1% BSA in PBS (2 h) and incubated with a 1 : 50 dilution of anti-DRAK2 antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) overnight at 41C. After serial washes, the slides were incubated with a 1 : 250 dilution of fluorescently labelled donkey anti-goat Alexa Fluor 488 (Molecular Probes, Leiden, the Netherlands) and subsequently propidium iodide (Molecular Probes) diluted at 1 mg ml À1 in PBS as a nuclear counterstain for 5 min. Slides were mounted with fluorescent mounting media (Dako, Carpinteria, CA, USA) and imaged with an LSM510 Zeiss Axioplan-2 upright confocal microscope (Carl Zeiss, Göttingen, Germany).