Fig. 4: YAP is actively imported into the nucleus by the nuclear transport receptor Imp7.
From: Mechanical control of nuclear import by Importin-7 is regulated by its dominant cargo YAP
a Association of endogenous Imp7 and YAP was analyzed by in situ PLA (red puncta) in RPE-1 cells using anti-YAP (63.7) and anti-Imp7 (rabbit) antibodies. Different controls are shown: anti-YAP or anti-Imp7 alone, and anti-YAP and anti-Imp7 with an unrelated antibody (anti-Impα5 and anti-MMP2, respectively). Images representative from three biologically independent experiments. b Lysates from RPE-1 cells grown at low confluence were immunoprecipitated with anti-Imp7 antibody. Immunoprecipitates and whole-cell lysates (WCL) were analyzed by immunoblotting with anti-YAP antibody and Imp7 as indicated in the figure. N-sMASE antibody was used as an immunoprecipitation antibody control. Three independent experiments were carried out. c Direct binding of YAP, but not YAP ΔC, to Imp7. GFP8Q, GFP8Q-YAP, or GFP8Q-YAP ΔC and GST-Imp7 or GST resin were combined (+) as indicated. Coomassie staining shows a fraction of the initial mixes (input) and pull-downs after the washes (right side). Representative of three biologically independent experiments. d Partitioning of GFP8Q-YAP, GFP8Q-YAP ΔC, and GFP8Q into FG particles derived from scNup116 FG domain. RanQ69L was used to block the interaction between cargo and the NTR. The brightness of image scans was individually adjusted. The average of partition coefficients is indicated for each condition. Representative of three independent experiments. e, f Import into nuclei of digitonin-permeabilized HeLa cells was followed for 5 min in the presence of an energy-regenerating system (EGS), a Ran mix, and 0.7 µM cargo GFP8Q-YAP that was pre-complexed with Imp7. Atto647N-labeled MBP (G260C mutant) was added as a control for the identification of leaky nuclei (cells labeled with white asterisks). Phase contrast was adjusted individually. Quantification is shown in graph (f). N = 42 cells in YAP + Imp7 + Ran, N = 36 cells in YAP + Ran, and N = 40 cells in YAP + Imp7 condition, data from 5 independent experiments. Raw data are available in the Source Data file. Statistical analysis with a two-tailed unpaired t-test. Data represent mean ± s.e.m. Scale bar 10 µm.
