FIGURE 3.
p27RF-Rho modulates RhoA and RhoC activation via inhibition of the p27kip1 pathway. a, regulation of Rho proteins by p27RF-Rho. B16F0 cells expressing an empty vector (mock) or p27RF-Rho fused to a V5 tag (p27RF-V5, lanes 1 and 2) and B16F10 cells expressing a control shRNA (sh-LacZ, lane 3) or either one of two shRNA sequences targeted to the p27RF-Rho mRNA (sh-p27RF1 and sh-p27RF2, lanes 3–5) were used for the assay. The cell lysates were analyzed by Western blot using antibodies that recognize p27RF-Rho, RhoA, or RhoC. Active Rho proteins were pulled down using the Rhotekin fragment and analyzed similarly. The ratio of active to total protein is indicated below the blots (n = 3). b, subcellular localization of p27kip1 in B16F0 and B16F10 cells. After cells were lysed and separated into cytoplasmic and nuclear fractions, p27kip1 was detected by Western blot analysis. Tubulin and lamin B1 are markers for cytoplasmic and nuclear proteins, respectively. T, total lysate; C, cytoplasmic fraction; N, nuclear fraction. c, p27RF-Rho suppresses p27kip1 so as to inhibit activation of RhoA and RhoC. An empty vector (mock, lane 1), p27kip1 fused to an mVenus tag (mVkip1, lanes 2–4), and mVkip1 with additional empty vector (mVkip1-mock, lane 3) or mVkip1 with p27RF-Rho fused to an mCherry tag (mVkip1-p27RFmC, lane 4) were expressed in B16F10 cells. Total and active Rho proteins were analyzed as in a. The ratio of active to total protein is indicated below the blots (n = 3).