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. 2004 Mar 26;101(14):5164–5169. doi: 10.1073/pnas.0300084101

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Fig. 5. — (A) Expression of the DIG3 cDNA in tobacco BY-2 protoplasts inhibits nuclear import of a GUS VirD2 NLS fusion protein. Fourteen hours after electroporation, protoplasts were stained with 5-bromo-4-chloro-3-indolyl β-d-glucuronide (X-gluc) and visualized by using a phase-contrast microscope. (Upper) Electroporation of a GUS-VirD2 NLS fusion gene. (Lower) Coelectroporation of DIG3 and a GUS-VirD2 NLS fusion gene. (B) Percentage of transfected protoplasts with GUS activity localized exclusively in the nucleus. Approximately 100 X-glucstained protoplasts were scored for each sample, and each experiment was performed three times in molar ratios of 10:1 and 20:1. Coelectroporation controls, vector, and GUSVirD2 NLS or DIG3 and GUS-NIa were performed at a ratio of 20:1. □, GUS-VirD2 NLS; ▪, DIG3 + GUS-VirD2 NLS (10:1); , DIG3 + GUS-VirD2 NLS (20:1); , vector + GUS-VirD2 NLS (20:1); , GUS-NIa; , DIG3 + GUS-NIa; , GUS. (C) A serine residue near the VirD2 C-terminal NLS is involved in nuclear import. Tobacco BY-2 protoplasts were electroporated with either the GUS-VirD2 NLS constructions alone or with the DIG3 gene. X-gluc-stained protoplasts were scored for the percentage of protoplasts that showed exclusive nuclear GUS localization. At least 300 transfected protoplasts were examined for each sample. ▪, GUS-VirD2 NLS; , GUS-VirD2 NLS (Ser-394→Ala).

Inline graphic — (A) Expression of the DIG3 cDNA in tobacco BY-2 protoplasts inhibits nuclear import of a GUS VirD2 NLS fusion protein. Fourteen hours after electroporation, protoplasts were stained with 5-bromo-4-chloro-3-indolyl β-d-glucuronide (X-gluc) and visualized by using a phase-contrast microscope. (Upper) Electroporation of a GUS-VirD2 NLS fusion gene. (Lower) Coelectroporation of DIG3 and a GUS-VirD2 NLS fusion gene. (B) Percentage of transfected protoplasts with GUS activity localized exclusively in the nucleus. Approximately 100 X-glucstained protoplasts were scored for each sample, and each experiment was performed three times in molar ratios of 10:1 and 20:1. Coelectroporation controls, vector, and GUSVirD2 NLS or DIG3 and GUS-NIa were performed at a ratio of 20:1. □, GUS-VirD2 NLS; ▪, DIG3 + GUS-VirD2 NLS (10:1); , DIG3 + GUS-VirD2 NLS (20:1); , vector + GUS-VirD2 NLS (20:1); , GUS-NIa; , DIG3 + GUS-NIa; , GUS. (C) A serine residue near the VirD2 C-terminal NLS is involved in nuclear import. Tobacco BY-2 protoplasts were electroporated with either the GUS-VirD2 NLS constructions alone or with the DIG3 gene. X-gluc-stained protoplasts were scored for the percentage of protoplasts that showed exclusive nuclear GUS localization. At least 300 transfected protoplasts were examined for each sample. ▪, GUS-VirD2 NLS; , GUS-VirD2 NLS (Ser-394→Ala).