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. 2012 Apr 20;7(4):e35848. doi: 10.1371/journal.pone.0035848

Figure 3. pVHL mediates K63-linked ubiquitination of nCLU.

Figure 3

(A) pVHL enhanced the K63-linked ubiquitination of nCLU. 293T cells were transfected with Myc-nCLU, R-VHL and HA-Ub(K48R) (or Ub(K63R)). In 24 h, the cells were lysed and immunoprecipitation was done using HA antibody. The relative amount of ubiquitinated nCLU was determined by densitometry assay. (B) pVHL enhanced the ubiquitination of nCLU in the presence of Ub(K63). 293T cells were transfected with Myc-nCLU, R-VHL and HA-Ub(K63) plasmids. In 24 h, the cells were harvested and then lysed for immunoprecipitation using HA antibody. (C) pVHL did not enhance nCLU ubiquitination in the presence of Ub(K48). 293T cells were transfected with Myc-nCLU, R-VHL and HA-Ub(K48) plasmids. In 24 h, the cells were harvested and then lysed for immunoprecipitation using HA antibody. (D) pVHL(1-155) did not mediate ubiquitination of nCLU. 293T cells were transfected with various constructs as indicated. In 24 h, the cell lysates were prepared for immunoprecipitation and western-blot. (E) Overexpression of Rbx1 enhanced K63-linked ubiquitination of nCLU. 293T cells were transfected as indicated. In 24 h, the cells were harvested and cell lysates were prepared for immunoprecipitation. The relative amount of ubiquitinated Myc-nCLU was determined by densitometry assay. (F) pVHL mediated K63-linked ubiquitination of nCLU in vitro. In vitro ubiquitination assay was performed as described under Methods.