Figure 3. Loss of Hippo signaling enhances the cytoplasmic retention of Skp2.
(A and B) Immunoblot analysis of p27, Skp2, α-tubulin or PARP in the cytoplasmic (c) and nuclear (n) fractions of WT, Mst1/2 DKO (A) or Yap Tg (B) liver tissues.
(C and D) IHC staining (C) or immunoblot analysis of the cell fractions (D) of Skp2 and p27 in liver tissues from WT, Mst1/2 DKO, Mst1f/fMst2f/fYap+/flAlb-Cre (DKO Yap+/−) or Yap Tg mice. Scale bars: 20 µm.
(E) Immunofluorescent staining of Skp2 (red) and the nuclear counterstain (DAPI, blue) in primary MEFs isolated from WT, Lats1/2 KO (left panel) or Yap Tg (right panel) mice. Scale bars: 10 µm.
(F–H) Immunoblot analysis of acetylated (ace-) Skp2, Skp2,p-Yap, Yap, Mst1, Mst2 and GAPDH in the immunoprecipitates or total lysates of hepatocytes isolated from WT, Mst1/2 DKO (F), Yap Tg (G), or Yap KO (H) mice. For the detection of ace-Skp2, the loading of the immunoprecipitates was normalized according to the levels of total Skp2.
(I and J) Immunoassay assessing the ubiquitination of p27 (detected with anti-Myc) in the lysates of 293T cells expressing various combinations of Myc-tagged ubiquitin, Flag-tagged p27, HA-tagged Yap and HA-tagged WT Skp2 (I) or an acetylation-mimetic (KLKL) mutant form of Skp2 (J) as indicated.
(K) Immunoblot analysis of Skp2, p27 and GAPDH in liver tissues from WT and Skp2 KO mice infected with Ad-GFP, Ad-Skp2 (WT) or Ad-Skp2 (KLKL).
(L) A proposed working model for Yap activation regulating p27 stability through modulating Skp2 acetylation and sub-cellular localization.
See also Figure S3.