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Fig. 3. CapG nuclear import requires energy and importin-ß. (A) Western blot: recombinant Myc-CapG-V5-His₆ was probed with anti-myc, anti-V5 or anti-CapG antibodies. (B-D) CapG-V5 import in the absence of RRL and energy (B), in the presence of energy but without RRL (C), or in the presence of GTP--S and RRL (D). Staining was performed with anti-V5 antibody. B'-D', DAPI stain. (E-E') Nuclear import of NLS-TRITC-BSA in the presence of RRL and GTP--S. (F,G) Import of CapG-V5 (F) and NLS-TRITC-BSA (G) in the presence of energy and RRL. (F'-G') DAPI stain. (H,I) Negative controls: anti-V5 antibody staining in the absence of CapG-V5 (H) and TRITC-BSA lacking the SV40 NLS (I). (H'-I') DAPI stain. (J) Importin-ß is required for CapG import. J1, CapG localization; J2, importin-ß localization; J3, merged image of J1 and J2; J4, DAPI stain. Note that CapG localizes to the nucleus only in cells that do not react with anti-importin-ß antibodies. (K) Anti-importin-ß antibody prevents NLS-TRITC-BSA nuclear import. (K') DAPI stain. The SV40 large T-antigen NLS peptide did not inhibit nuclear accumulation of CapG (L). (L') DAPI stain. Bar, 50 µm.

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