pVHL Mediates Ubiquitination of nCLU Figure 4. pVHL enhances nuclear translocation of nCLU. (A) Overexpression of Ub(K63) promoted nCLU nucleus translocation. 293T cells were transfected with the constructs as indicated. In 24 h, the nuclear and cytoplasmic proteins were prepared for immunoblotting. (B) Overexpression of Ub(K63) promoted nCLU nucleus translocation. Hela cells were transfected with the constructs as indicated. In 24 h, the cells were fixed and immunostained as described under methods. The pictures were obtained under a confocal microscope. (C) Overexpression of pVHL promoted nCLU nuclear translocation. 293T cells were transfected as indicated. In 24 h, the cells were harvested and nuclear and cytoplasmic proteins were prepared for immunoblotting. (D) pVHL promoted nCLU nuclear translocation. Hela cells were transfected as indicated. In 24 h, the cells were fixed and immuno-stained as described in Methods. doi:10.1371/journal.pone.0035848.g004 Supporting Information Figure S1 (A) 293T cells were transfected with Myc-nCLU plus empty vector or Myc-nCLU plus HA-VHL. In 24 h, the cells were harvested and cellular proteins were prepared. Immunoprecipita- tion was performed using HA antibody. The results of negative control experiment showed that HA antibody did not interact with Myc-tagged nCLU. (B) 293T cells were transfected as indicated. In 24 h, the cells were harvested and immunoprecipitation was performed using HA antibody. The results of negative control experiment indicated that the HA antibody did not interact with Myc-tagged VHL. (TIF) Figure S2 pVHL(1-155) and pVHL(D63-155) had little effect on ubiquitination of nCLU. 293T cells were transfected References 1. Frew IJ, Krek W (2008) pVHL: a multipurpose adaptor protein. Sci Signal 1: e30. 2. Kim WY, Kaelin WG (2004) Role of VHL gene mutation in human cancer. J Clin Oncol 22: 4991–5004. 3. Kaelin WG (2007) Von Hippel-Lindau disease. Annu Rev Pathol 2: 145–173. with HA-nCLU, His-Ub and various Myc-VHL constructs as indicated. In 24 h, the cells were harvested and cellular proteins were prepared for immunoprecipitation and western-blot. (TIF) Figure S3 pVHL mediated K63-linked ubiquitination of nCLU in vitro. In vitro ubiquitination of nCLU by pVHL was performed as described under Methods. Ubiquitin or Ub(K63R) at 5 mg/mL was added to the reaction system. (TIF) Author Contributions Conceived and designed the experiments: JF. Performed the experiments: JX DL SJ WZ HS XL BY LF. Contributed reagents/materials/analysis tools: RH. Wrote the paper: JF. 4. Kamura T, Koepp DM, Conrad MN, Skowyra D, Moreland RJ, et al. (1999) Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase. Science 284(5414): 657–61. 5. Iwai K, Yamanaka K, Kamura T, Minato N, Conaway RC, et al. (1999) Identification of the von Hippel-lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex. Proc Natl Acad Sci USA 96: 12436–12441. PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35848