Fig. 2.

WWOX binds c-Jun and prevents its nuclear translocation. A549 cells were transfected with a FLAG-tagged WWOX expression construct, and a coimmunoprecipitation was performed using anti-FLAG antibody conjugated to magnetic beads in n = 3 experiments. A: representative results of the coimmunoprecipitation experiment. A mock immunoprecipitation (IP) was performed in A549 cells transfected with a control plasmid to ensure that resulting immunoprecipitation bands were not due to nonspecific protein binding to the beads. The Western blot shown here illustrates pulldown of c-Jun along with FLAG-WWOX. Note that since WWOX and c-Jun are of similar molecular weight, the bands for each are derived from separate gels that were run at the same time and that contained the same fractions of the same eluents, supernatants, and lysates. In separate experiments, A549 cells were cotransfected with a HA-tagged c-Jun expression construct and either a control miR-GFP- or WWOX-silencing miR-GFP-expressing plasmid. Cells were then stained with anti-HA-Alexa Fluor 594-conjugated antibody (red) along with DAPI counterstain. B: confocal microscopic images illustrate increased nuclear translocation of c-Jun during WWOX silencing vs. control observed in n = 3 experiments. Cells in 10 high-power fields (hpf) were counted and the percentage showing strong nuclear staining are depicted in the accompanying bar graph. C: these results were confirmed by Western blotting of nuclear (nuc) vs. cytoplasmic (cyt) fractions derived from cells transfected with control vs. WWOX-silencing miR-expressing plasmids in n = 3 experiments. *P < 0.05, significantly different from control by Student’s t-test.