Fig. 3. Mutant NAF1 lacks a C-terminal NLS.

(A) NAF1 amino acid alignment within and flanking the NLS for disease-associated mutations and those studied for nuclear localization by immunofluorescence are additionally shown. The four conserved lysine residues are indicated by dots above, and the bipartite NLS sequences are underlined. (B) Western blot of Myc-tagged NAF1 in the HeLa doxycycline-inducible system after 3-hour exposure to leptomycin B. (C) Immunofluorescence images showing subcellular localization of Myc-tagged NAF1 (green). Fluorophores in the three-color overlay are labeled to the left. Images were taken at ×63 magnification (scale bar, 20 µm). DAPI, 4′,6-diamidino-2-phenylindole. (D) Quantification of the NAF1 cytoplasmic fraction represents the intensity of cytoplasmic anti-Myc staining (defined by tubulin-positive area) relative to the nuclear staining (DAPI area). About 100 cells were quantified for each cell line, and mean values are shown with error bars representing SEM. Analysis was performed blinded to genotype. (E)Western blot of total (T), cytoplasmic (C), and nuclear (N) protein lysates of parental HCT116 and CRISPR/Cas9-edited HCT116 cells. For the quantification shown below, the total and cytoplasmic fractions were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH), whereas the nuclear fraction was normalized to poly(ADP-ribose) polymerase (PARP). ***P < 0.001 (Student’s t test).