For immunofluorescence CRFK cells were seeded onto 19 mm coverslips in 6 well trays and were transfected with RNA as described. Following a 24 hr incubation the coverslips were washed with PBS and fixed in 4% formaldehyde for 15 min at room temperature. Cells were permeabilised and blocked in saponin buffer, also used as staining buffer, (0.1% saponin, 10% foetal calf serum, 0.1% sodium azide) for 1 hr at 4uC. Cells were stained using an anti-V5 monoclonal antibody (Invitrogen) followed by an anti-mouse Alexafluor 488 conjugated secondary antibody (Molecular Probes) at the recommended dilution in staining buffer for 30 min in the dark. Cells were then stained for 30 min in the dark with a Wheat Germ Agglutinin Alexafluor 594 nm conjugate (Molcular Probes) to allow identification of plasma and Golgi membranes. Coverslips were washed and mounted onto slides using Vectashield containing DAPI (Vector Labs). Microscopy was performed using an inverted Leica TCS-NT confocal laser scanning microscope.