Figure 3.
The WW domain is identified as a novel class of folded NLS. (A) Analysis of the transcriptional activities of YAP1 and YAP1 truncations in HEK293T cells (representative of three independent experiments, N = 3, all data shown as the average values ± SD, two-tailed Student’s t test). The representative western blotting shows expressions of YAP1 and the truncations used in reporter assays. (B) The table and schematic diagram show the amino acid sequences of WW domains derived from different proteins. (C and D) Representative images show subcellular distributions of GFP fused with tdWWs derived from different donor proteins in HeLa cells. The inserted curve charts show relative fluorescent intensity of GFP and Hoechst staining along the orange indicator lines with the arrow, which across the cytoplasm and nucleus (collected using LAS AF Lite software) (C). The scatter chart shows nucleocytoplasmic ratios of GFP fused with different WW domains (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test) (D). (E) The transcriptional activity of the YAP1-ΔWWs truncated mutant is rescued by the heterologous WW domains derived from WWP2 or PIN1 in HEK293T cells, respectively (representative of three independent experiments, N = 3, all data shown as the average values ± SD, two-tailed Student’s t test). (F) The nuclear localization of the YAP1-ΔWWs mutant is rescued by the heterologous WW domains derived from PIN1 in HEK293T cells (the cyan line indicates the nuclear outline). The scatter chart shows nucleocytoplasmic ratios of YAP1 and the mutants (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test). (G) Representative immunostaining images show subcellular distributions of GSK3β-FLAG and the 2xWW (PIN1)−GSK3β-FLAG in HeLa cells. (H) Representative images show subcellular distributions of the tdWWs (YAP1)−3xGFP and 2xWW (WWP2)−3xGFP in the absence or presence of ectopic RAN-Q69L mutant in HEK293T cells (the yellow line indicates the outline of the nucleus), and the scatter chart shows their nucleocytoplasmic ratios (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test). Source data are available for this figure: SourceData F3.

The WW domain is identified as a novel class of folded NLS. (A) Analysis of the transcriptional activities of YAP1 and YAP1 truncations in HEK293T cells (representative of three independent experiments, N = 3, all data shown as the average values ± SD, two-tailed Student’s t test). The representative western blotting shows expressions of YAP1 and the truncations used in reporter assays. (B) The table and schematic diagram show the amino acid sequences of WW domains derived from different proteins. (C and D) Representative images show subcellular distributions of GFP fused with tdWWs derived from different donor proteins in HeLa cells. The inserted curve charts show relative fluorescent intensity of GFP and Hoechst staining along the orange indicator lines with the arrow, which across the cytoplasm and nucleus (collected using LAS AF Lite software) (C). The scatter chart shows nucleocytoplasmic ratios of GFP fused with different WW domains (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test) (D). (E) The transcriptional activity of the YAP1-ΔWWs truncated mutant is rescued by the heterologous WW domains derived from WWP2 or PIN1 in HEK293T cells, respectively (representative of three independent experiments, N = 3, all data shown as the average values ± SD, two-tailed Student’s t test). (F) The nuclear localization of the YAP1-ΔWWs mutant is rescued by the heterologous WW domains derived from PIN1 in HEK293T cells (the cyan line indicates the nuclear outline). The scatter chart shows nucleocytoplasmic ratios of YAP1 and the mutants (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test). (G) Representative immunostaining images show subcellular distributions of GSK3β-FLAG and the 2xWW (PIN1)−GSK3β-FLAG in HeLa cells. (H) Representative images show subcellular distributions of the tdWWs (YAP1)−3xGFP and 2xWW (WWP2)−3xGFP in the absence or presence of ectopic RAN-Q69L mutant in HEK293T cells (the yellow line indicates the outline of the nucleus), and the scatter chart shows their nucleocytoplasmic ratios (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test). Source data are available for this figure: SourceData F3.

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