Figure 4

Transportin-2 is required for PTEN translocation to the nucleus. (A) Transportin-2 protein is inhibited by vmr11: Uninfected PPH cultures were transfected with 50 nM oligonucleotides (from left to right): scrambled vmr11 (Ctl), vmr11 mimic (vmr11), vmr11 mimic plus vmr11-antagomir (vmr11-Ctl), TRN-2 siRNA (Si-TRN-2), scrambled siTRN-2 (Si-Ctl) and siTRN-2 plus TRN-2 cDNA expression vector (Si-TRN-2 + TRN2-CFP). Western blots of TRN-2 protein from cells harvested 48 hours post-transfection were quantitated from three independent assays using β-Actin as loading control. (B) Nuclear PTEN is restricted by vmr11 or siTRN-2 knock-down: PPH cultures were transfected with 50 nM oligonucleotides (from left to right): scrambled vmr11 (ctrl), vmr11 “mimic” (vmr11), si-TRN2, scrambled si-TRN2 (si-ctl) or 1 μg TRN-2 cDNA expression vector following TRN-2 knock-down (si-TRN2 + TRN2-CFP). Transfections were repeated twice at 24 hour intervals and cells were harvested at 48 hour post-transfection; Western blot of nuclear PTEN was quantitated (with Lamin B1 as loading control); results shown are from three independent assays. (C) Transportin-2-PTEN protein interaction: Immunoprecipitates with PTEN antibody or IgG controls were analyzed by Western blot for PTEN or TRN-2 protein. PTEN-TRN-2 protein interaction is shown in the far right lane. (D) Suboptimal PTEN-Transportin-2 protein complex is restricted at nuclear membrane: Human primary hepatocytes were transfected with vmr11, Si-TRN2 and Si-Ctl oligonucleotides (50 nM of vmr11 mimic, twice at 0 hour and 24 hour). Cells were processed at 48 hrs post-transfection. PTEN is stained with FITC and TRN-2 with Texas Red.