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. 2016 Apr 20;7(19):26979–26991. doi: 10.18632/oncotarget.8876

Figure 4. The membrane-associated Ccnd1 promotes Ral-GTPase activation.

Figure 4

A. HEK293T cells were co-transfected with HA-RalB and either HA-Ccnd1, or HA-Ccnd1-CAAX or an empty vector as a negative control. Twenty-four hours after transfection, active RalB-GTP was affinity purified with RalBP-beads from cell lysates and detected by immunoblotting with anti-HA antibody. HA-D1, HA-RalB and HA-RalB-GTP amount from a representative experiment is shown. RalBP-beads and actin were used as loading controls. B. The experiment was independently repeated four times. Relative mean values ± sem for the HA-RalB-GTP/ total HA-RalB ratio are plotted. Significance values were determined by one way ANOVA and Holm Τ-statistic post-test (*p < 0.05; **p < 0.01). C. R3327-5′A rat cells were infected with interference shRNA against RalB (shRalB) or with scramble (scr) as a control. Relative invasion values are expressed in mean ± sem. Data are from three independent experiments. Significance values were determined by one way ANOVA and Holm Τ-statistic post-test (**p < 0.01). D. Immunoblot showing the levels of expression of RalB in (C). Gel stain was used as a loading control.