Figure S2.
The WW domain is identified as a novel class of folded NLS. (A) Representative immunostaining images show subcellular distributions of β-tubulin-FLAG and the 2xWW (PIN1)−β-tubulin-FLAG in HeLa cells. (B) Representative images show subcellular distributions of 3xGFP proteins fused with the wild-type WW domain or the mutant WW domain bearing the substitutions of two conserved tryptophan residues (AA) in HeLa cells. The WW domain is derived from WWP2. (C) Examination of the efflux rates of GFP and 2xWW (YAP1)−GFP from the nucleus after digitonin (40 μg/ml) permeabilization, the nuclear content of c-JUN as a control for the integrity of the nuclear envelope. The monoclonal HeLa cells with the uniform expression of GFP or 2xWW (YAP1)−GFP are employed to carry out the experiment. The WW domain is derived from the second WW domain of human YAP1. The relative nuclear contents of GFP or 2xWW (YAP1)−GFP at 0, 2, 6, and 10 min after permeabilization are plotted in a violin chart (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test). (D) Representative images show the subcellular localization of 2xWW (PIN1)−GFP in the absence or presence of RAN-Q69L (the yellow line indicates the cell boundary and the magenta 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). (E) Data from Uniprot, Simple Modular Architecture Research Tool, and the Human Protein Atlas shows subcellular localization of human WW-containing proteins. Number, the number of proteins in each category. (F) Representative immunostaining images show subcellular distributions of the wild-type TAZ, the TAZ-ΔWW mutant, the wild-type PIN1, and the PIN1-ΔWW mutant in HEK293T cells (the cyan line indicates the nuclear outline). (G) Representative immunostaining images show subcellular distributions of the wild-type KIBRA and the KIBRA-ΔWW mutant in HEK293T cells. LMB (0.5 μM) is applied for 2 h. (H) The WW domain regulates the transcriptional activity of TAZ 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 TAZ and the mutant. Source data are available for this figure: SourceData FS2.

The WW domain is identified as a novel class of folded NLS. (A) Representative immunostaining images show subcellular distributions of β-tubulin-FLAG and the 2xWW (PIN1)−β-tubulin-FLAG in HeLa cells. (B) Representative images show subcellular distributions of 3xGFP proteins fused with the wild-type WW domain or the mutant WW domain bearing the substitutions of two conserved tryptophan residues (AA) in HeLa cells. The WW domain is derived from WWP2. (C) Examination of the efflux rates of GFP and 2xWW (YAP1)−GFP from the nucleus after digitonin (40 μg/ml) permeabilization, the nuclear content of c-JUN as a control for the integrity of the nuclear envelope. The monoclonal HeLa cells with the uniform expression of GFP or 2xWW (YAP1)−GFP are employed to carry out the experiment. The WW domain is derived from the second WW domain of human YAP1. The relative nuclear contents of GFP or 2xWW (YAP1)−GFP at 0, 2, 6, and 10 min after permeabilization are plotted in a violin chart (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test). (D) Representative images show the subcellular localization of 2xWW (PIN1)−GFP in the absence or presence of RAN-Q69L (the yellow line indicates the cell boundary and the magenta 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). (E) Data from Uniprot, Simple Modular Architecture Research Tool, and the Human Protein Atlas shows subcellular localization of human WW-containing proteins. Number, the number of proteins in each category. (F) Representative immunostaining images show subcellular distributions of the wild-type TAZ, the TAZ-ΔWW mutant, the wild-type PIN1, and the PIN1-ΔWW mutant in HEK293T cells (the cyan line indicates the nuclear outline). (G) Representative immunostaining images show subcellular distributions of the wild-type KIBRA and the KIBRA-ΔWW mutant in HEK293T cells. LMB (0.5 μM) is applied for 2 h. (H) The WW domain regulates the transcriptional activity of TAZ 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 TAZ and the mutant. Source data are available for this figure: SourceData FS2.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal