Fusion of conserved NES protein motifs to AF10 confers cytoplasmic localization and in vitro immortalization potential. (A) Alignment of the CALM NES (aa 540-557) and the NES motifs from heterologous proteins, ABL1 (aa 1086-1103), Rev (aa 70-87), APC (aa 63-79), and PKIA (aa 33-50), fused in-frame with AF10 (at aa 234). Key hydrophobic residues within each NES are highlighted. (B) Confocal IF analysis of HEK293 cells transfected with Flag-tagged AF10 (aa 234-1068) or the NES^CALM-AF10 fusion in the absence (middle) or presence (right) of LMB (10 nM, 1 h). (C) Confocal IF of HEK293 cells transfected with the heterologous NES-AF10 fusions. Cell nuclei were stained with DAPI (blue). Bars represent 20 μm. (D) Colony-forming assay of the NES-AF10 constructs. Bars represent the number of colonies generated per 10 000 cells seeded in second and third round cultures. The mean ± SEM are shown from duplicate samples analyzed in 3 (NES^ABL1-AF10), 4 (NES^CALM-AF10, NES^PKIA-AF10, NES^APC-AF10, NES^Rev-AF10), or 5 (Vector and CALM-AF10) independent experiments.