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. Author manuscript; available in PMC: 2014 Nov 1.

Published in final edited form as: Virology. 2013 Sep 10;446(0):334–345. doi: 10.1016/j.virol.2013.08.017

Fig. 5 — A. Mutation of hydrophobic residues within the zinc-binding domain disrupts the nuclear localization of 2xEGFP-16E7. HeLa cells were transfected with either 2xEGFP-16E7 wild type (panels A, E, and I), 2xEGFP-16E7_LRLCV65AAAAA (panels B, F, and J), 2xEGFP-16E7_R66A (panels C, G, and K), or 2xEGFP (panels D, H, and L) plasmids and examined by confocal fluorescence microscopy at 24 h post-transfection. Panels A-D represent DAPI staining of the nuclei, panels E-H represent EGFP fluorescence, and panels I-L represent the merge. B. Quantitative analysis of the effect of mutation of hydrophobic residues within the zinc-binding domain on the localization of 2xEGFP-16E7 in HeLa cells. Data from seven experiments using 2xEGFP-16E7 wild type, 2xEGFP-16E7_LRLCV65AAAAA, 2xEGFP-16E7_R66A, and 2xEGFP plasmids were used for quantitative analysis and graphic representation of the localization distribution of transfected HeLa cells. Black bars represent cells exhibiting predominant nuclear localization; gray bars represent cells exhibiting pancellular localization; white bars represent cells exhibiting predominant cytoplasmic localization. C. Quantitative analysis of the effect of mutation of hydrophobic residues within the zinc-binding domain on the localization of EGFP-16E7 in HeLa cells. Data from five experiments using EGFP-16E7 wild type, EGFP-16E7_LRLCV65AAAAA, EGFP-16E7_R66A, and EGFP plasmids were used for quantitative analysis and graphic representation of the localization distribution of transfected HeLa cells. Black bars represent cells exhibiting predominant nuclear localization; gray bars represent cells exhibiting pancellular localization; white bars represent cells exhibiting predominant cytoplasmic localization. D. EGFP-16E7 and 2xEGFP-16E7 hydrophobic residues and R66A mutants are properly expressed in HeLa cells. HeLa cells were transfected with 2xEGFP-16E7 (lane 1), 2xEGFP-16E7_LRLCV65AAAAA (lane 2), 2xEGFP-16E7_R66A (lane 3), 2xEGFP (lane 4), EGFP-16E7 (lane 5), EGFP-16E7_LRLCV65AAAAA (lane 6), EGFP-16E7_R66A (lane 7), or EGFP (lane 8) plasmids. Cell lysates were prepared 24 h post transfection and probed with a GFP antibody.

Fig. 5 — A. Mutation of hydrophobic residues within the zinc-binding domain disrupts the nuclear localization of 2xEGFP-16E7. HeLa cells were transfected with either 2xEGFP-16E7 wild type (panels A, E, and I), 2xEGFP-16E7_LRLCV65AAAAA (panels B, F, and J), 2xEGFP-16E7_R66A (panels C, G, and K), or 2xEGFP (panels D, H, and L) plasmids and examined by confocal fluorescence microscopy at 24 h post-transfection. Panels A-D represent DAPI staining of the nuclei, panels E-H represent EGFP fluorescence, and panels I-L represent the merge. B. Quantitative analysis of the effect of mutation of hydrophobic residues within the zinc-binding domain on the localization of 2xEGFP-16E7 in HeLa cells. Data from seven experiments using 2xEGFP-16E7 wild type, 2xEGFP-16E7_LRLCV65AAAAA, 2xEGFP-16E7_R66A, and 2xEGFP plasmids were used for quantitative analysis and graphic representation of the localization distribution of transfected HeLa cells. Black bars represent cells exhibiting predominant nuclear localization; gray bars represent cells exhibiting pancellular localization; white bars represent cells exhibiting predominant cytoplasmic localization. C. Quantitative analysis of the effect of mutation of hydrophobic residues within the zinc-binding domain on the localization of EGFP-16E7 in HeLa cells. Data from five experiments using EGFP-16E7 wild type, EGFP-16E7_LRLCV65AAAAA, EGFP-16E7_R66A, and EGFP plasmids were used for quantitative analysis and graphic representation of the localization distribution of transfected HeLa cells. Black bars represent cells exhibiting predominant nuclear localization; gray bars represent cells exhibiting pancellular localization; white bars represent cells exhibiting predominant cytoplasmic localization. D. EGFP-16E7 and 2xEGFP-16E7 hydrophobic residues and R66A mutants are properly expressed in HeLa cells. HeLa cells were transfected with 2xEGFP-16E7 (lane 1), 2xEGFP-16E7_LRLCV65AAAAA (lane 2), 2xEGFP-16E7_R66A (lane 3), 2xEGFP (lane 4), EGFP-16E7 (lane 5), EGFP-16E7_LRLCV65AAAAA (lane 6), EGFP-16E7_R66A (lane 7), or EGFP (lane 8) plasmids. Cell lysates were prepared 24 h post transfection and probed with a GFP antibody.