Cyclin A2 directly interacts with RhoA and regulates its activation state. (A–C, top) Western blotting after pull-down of activated forms of RhoA (A), Rac1 (B), or RhoC (C) after depletion (A, left–C) or forced expression (A, right) of Cyclin A2. (bottom) Corresponding quantifications from independent pull-down experiments (normalization of the GTP-bound forms to total RhoA, Rac1, or RhoC and GAPDH). Data are represented as means ± SEM (*, P = 0.031; **, P = 0.003; n = 5 for shCycA2, n = 3 for CycA2-Flag for the RhoA pull-down assay, and n = 3 for the Rac1 and RhoC pull-down assays). (D) Western blot analysis (top) and quantification (bottom) of the phosphorylation state of the RhoA–ROCK effector Cofilin (±SEM; *, P = 0.031; n = 3). (E) Detection of the rescue of cytoskeleton corticalization by constitutively active RhoA (green) in Cyclin A2–depleted cells using F-Actin staining (red). The arrow points to a RhoA-expressing cell, and the arrowhead points to a nontransfected cell. Bars, 20 µm (F) Coimmunoprecipitation (IP) of endogenous Cyclin A2 with RhoA. Cell lysates obtained from NIH3T3 cells transfected with RhoAV14-GFP were immunoprecipitated by GFP-Trap beads. Interaction between the two molecules was detected after blotting with an anti–Cyclin A2 antibody after GFP pull-down. (G) Cyclin A2 directly interacts with RhoA. Recombinant Cyclin A2 was incubated either with glutathione beads as control or RhoA-GST bound to glutathione beads. Cyclin A2 bound to RhoA-GST was detected by Western blotting after elution. Molecular markers are given in kilodaltons.