Fig 1: Cortical recruitment of caveolin-1 depends on the RhoA signalling pathway.(a) Western blot analysis of mDia1, Ect2, RhoA, caveolin-1 and α-tubulin (control) in the cells in b. (b) Three-dimensional reconstituted images of prometaphase cells and metaphase cells with cell rounding defects, which were treated with two independent RhoA siRNAs, Ect2 siRNAs, mDia1 siRNAs, or control luci siRNA, and stained with an anti-caveolin-1 antibody (green) and Hoechst (blue). The region with a caveolin-1 intensity higher than a certain threshold is represented as the enclosed red area in the left panel. (c) Average of the integrated caveolin-1 intensity in the red region in b was measured using z-stack images for each sample (mean±s.d. from three independent experiments; n>20/experiment) (d) Three-dimensional reconstituted images of prometaphase cells treated with or without a Rho inhibitor or activator and stained with an anti-caveolin-1 antibody (green) and Hoechst (blue). The region with the caveolin-1 intensity higher than a certain threshold is represented as the enclosed red area in the left panel. (e) Average of the integrated caveolin-1 intensity in the red region in d was measured using z-stack images for each sample (mean±s.d. from three independent experiments; n>20/experiment). (f) Total lysates from mitotically arrested cells expressing GFP, GFP-caveolin-1, or GFP-LGN were subjected to immunoprecipitation with an anti-GFP antibody and subjected to western blotting with anti-GFP, anti-RhoA and anti-Gαi1 antibodies. (g) Co-immunoprecipitation analysis of exogenously expressed HA-RhoA wt, HA-RhoA V14 (constitutively active form), or RhoA N19 (dominant negative form) with GFP-caveolin-1 in mitotically arrested cells. (h) Co-immunoprecipitation analysis of endogenous RhoA or Gαi1 with GFP-caveolin-1 or control GFP in synchronized cells released from the double thymidine block. *P<0.05 and ***P<0.001, analysed by Dunnett's multiple-comparison test.