Figure 5.
KPNA−KPNB1 axis directs YAP1 nuclear import. (A) The schematic diagram illustrates the in vitro binding assays of the YAP1/KPNA2 complex. Fc-fused YAP1 is overexpressed in HEK293T cells and then collected by protein A/G plus agarose. For binding assays, the recombinant His-KPNA2 proteins (3.3 μM) obtained from E. coli are incubated with A/G agarose engaging Fc−YAP1 for 1 h at 4°C, and the Fc fragment as the control. After washing, the pulled-down protein complexes by A/G agarose were analyzed using SDS PAGE. (B) In vitro binding assays of recombinant His-KPNA2 and His-xPxAA mutant of KPNA2 with Fc−YAP1 and Fc−W4A (YAP1), respectively. (C) In vitro binding assays of recombinant His-KPNA2 with Fc−WW (the second WW domain of YAP1). (D) Representative images show subcellular distributions of YAP1-GFP following 2-h LMB (0.5 μM) treatment in HEK293T cells bearing transient knockdown of KPNB1 expression. The scatter chart shows YAP1-GFP nucleocytoplasmic ratios (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test), and the representative western blotting shows KPNB1 contents in scramble control and KPNB1-knockdown HEK293T cells. (E) In vitro binding assays of recombinant His-KPNA2 and the IBB deletion mutant ΔIBB with Fc−YAP1, respectively. The inserted schematic diagrams illustrate binding sites for KPNB1, cNLS, and WW domain on KPNA (lower panel) and the hypothesis of IBB-facilitated release of WW cargoes from KPNAs (upper panel). Source data are available for this figure: SourceData F5.

KPNA−KPNB1 axis directs YAP1 nuclear import. (A) The schematic diagram illustrates the in vitro binding assays of the YAP1/KPNA2 complex. Fc-fused YAP1 is overexpressed in HEK293T cells and then collected by protein A/G plus agarose. For binding assays, the recombinant His-KPNA2 proteins (3.3 μM) obtained from E. coli are incubated with A/G agarose engaging Fc−YAP1 for 1 h at 4°C, and the Fc fragment as the control. After washing, the pulled-down protein complexes by A/G agarose were analyzed using SDS PAGE. (B) In vitro binding assays of recombinant His-KPNA2 and His-xPxAA mutant of KPNA2 with Fc−YAP1 and Fc−W4A (YAP1), respectively. (C) In vitro binding assays of recombinant His-KPNA2 with Fc−WW (the second WW domain of YAP1). (D) Representative images show subcellular distributions of YAP1-GFP following 2-h LMB (0.5 μM) treatment in HEK293T cells bearing transient knockdown of KPNB1 expression. The scatter chart shows YAP1-GFP nucleocytoplasmic ratios (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test), and the representative western blotting shows KPNB1 contents in scramble control and KPNB1-knockdown HEK293T cells. (E) In vitro binding assays of recombinant His-KPNA2 and the IBB deletion mutant ΔIBB with Fc−YAP1, respectively. The inserted schematic diagrams illustrate binding sites for KPNB1, cNLS, and WW domain on KPNA (lower panel) and the hypothesis of IBB-facilitated release of WW cargoes from KPNAs (upper panel). Source data are available for this figure: SourceData F5.

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