![Figure 2. CALM contains a functional NES that is necessary for CALM-AF10–mediated immortalization. (A) Alignment of the NES within CALM (aa 544-553) and the consensus sequence of CRM1-dependent NES, where ϕ represents any hydrophobic residue and x represents any amino acid. The hydrophobic aa of the CALM NES were point-mutated to alanines (A) to create 3 mutants: CALM(mutNES1)-AF10, CALM(mutNES2)-AF10, and CALM(mutNES1+2)-AF10. (B) Confocal immunofluorescence (IF) analysis of HEK293 cells transiently transfected with Flag-tagged CALM-AF10 and analyzed in the absence (left) or presence (right) of LMB (10 nM, 1 h). (C) Confocal IF analysis of murine CalmNULL CALM-AF10 leukemia cells grown in the absence (left) or presence (right) of LMB (0.1 nM, 12 h). (D) Confocal IF of HEK293 cells transiently transfected with CALM(mutNES)-AF10 mutants. (E) Confocal IF of MLL-ENL–immortalized CalmNULL hematopoietic precursors retrovirally infected with Flag-tagged CALM-AF10 (left panels) or CALM(mutNES1+2)-AF10 (right panels). Cell nuclei were visualized with DAPI (blue). Bars represent 20 μm for all panels. (F) Colony-forming assay of murine HPs infected with empty vector, CALM-AF10, or NES point mutants. Bars represent the number of colonies generated per 10 000 cells seeded in second and third passage cultures. The mean ± SEM are shown from duplicate samples analyzed in 2 [CALM(mutNES1)-AF10], 3 [CALM(mutNES2)-AF10], and CALM[mutNES1+2)-AF10] or 6 (Vector and CALM-AF10) independent experiments. Bottom panel is a western blot of HEK293 cells transfected with empty vector, CALM-AF10, and CALM(mutNES)-AF10 point mutants.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/121/23/10.1182_blood-2012-06-435792/4/4758f2.jpeg?Expires=1723728296&Signature=1qt68~pGqyupdXyYzyPobnPyLjPsagAQTb3vbgLIfLIo5pPvJeQgMwRogMtt57yvqnrCkheNkwSR7W3BIiTmzMfF8yrpVn1yV63Ts85W01GJBojZNBH3qrgvJRPQ3g3g0fQi~w1sZQzd2jEvl7vqTPJhrBdqB9jX0gKFj6--XFxGVo4w86nsWcHdrN6s1QhwUxiGpUFyfRz9rZLSsemwDOkV3AFrdmgg-0nADGtDk3~Mz3svauXsOnSGafRrfr0GZuhstKy-B4QPSDYgjZBDcSFh-fhU-NjgcjvsCf18nVsRUlUzxNep3n-~7eTozJjnF3Gv~UzInO~bVLpiZPEirA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CALM contains a functional NES that is necessary for CALM-AF10–mediated immortalization. (A) Alignment of the NES within CALM (aa 544-553) and the consensus sequence of CRM1-dependent NES, where ϕ represents any hydrophobic residue and x represents any amino acid. The hydrophobic aa of the CALM NES were point-mutated to alanines (A) to create 3 mutants: CALM(mutNES1)-AF10, CALM(mutNES2)-AF10, and CALM(mutNES1+2)-AF10. (B) Confocal immunofluorescence (IF) analysis of HEK293 cells transiently transfected with Flag-tagged CALM-AF10 and analyzed in the absence (left) or presence (right) of LMB (10 nM, 1 h). (C) Confocal IF analysis of murine CalmNULLCALM-AF10 leukemia cells grown in the absence (left) or presence (right) of LMB (0.1 nM, 12 h). (D) Confocal IF of HEK293 cells transiently transfected with CALM(mutNES)-AF10 mutants. (E) Confocal IF of MLL-ENL–immortalized CalmNULL hematopoietic precursors retrovirally infected with Flag-tagged CALM-AF10 (left panels) or CALM(mutNES1+2)-AF10 (right panels). Cell nuclei were visualized with DAPI (blue). Bars represent 20 μm for all panels. (F) Colony-forming assay of murine HPs infected with empty vector, CALM-AF10, or NES point mutants. Bars represent the number of colonies generated per 10 000 cells seeded in second and third passage cultures. The mean ± SEM are shown from duplicate samples analyzed in 2 [CALM(mutNES1)-AF10], 3 [CALM(mutNES2)-AF10], and CALM[mutNES1+2)-AF10] or 6 (Vector and CALM-AF10) independent experiments. Bottom panel is a western blot of HEK293 cells transfected with empty vector, CALM-AF10, and CALM(mutNES)-AF10 point mutants.
