Fig. 2.

MCM3AP and GANP have different localisations in the cell. (a) MCM3AP can shuttle between the cytoplasm and nucleus. 293 T cells were transfected with EYFP-MCM3AP plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Twenty-four hours after transfection, cells were treated with leptomycin B for 90 min and analysed by immunofluorescence as described previously.⁷ (b) MCM3AP localises to the nucleus and cytoplasm, whereas GANP localises to the nuclear envelope. MCM3AP and GANP stable cell lines were generated as follows. Full-length GANP or MCM3AP sequence was amplified by PCR, adding KpnI and XhoI restriction enzyme ends. The KpnI site was designed to create a Kozak sequence around the start codon in both cases. The PCR fragment was ligated into pUB6/V5-His A (Invitrogen) in the correct reading frame to add C-terminal V5 and poly-His tags. Then, KpnI and PmeI were used to excise the protein and tags to ligate into pcDNA5/FRT/To (Invitrogen), which had been mutated to remove an upstream PmeI site. This construct was co-transfected with pOG44 into Flp-In TREx 293 cells according to the manufacturer's protocol (Invitrogen). This after antibiotic selection produces a cell line that can be induced to express GANP or MCM3AP by the addition of 0.1 mg/ml doxycycline. GANP and MCM3AP localisations were determined by immunofluorescence with anti-V5 antibody (Invitrogen). (c) Expression of GANP-V5 and MCM3AP-V5 were validated by Western blot using anti-V5 antibody.