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. 2014 Dec 23;6(2):771–788. doi: 10.18632/oncotarget.2718

Figure 4. SKP2 regulates the ubiquitination of JARID1B through TRAF6.

Figure 4

(A) Immunofluorescence images show a co-localization of endogenous JARID1B and TRAF6 in PC3 cells. Scale bars represent 10 μm. (B) and (C) Co-immunoprecipitation analysis shows that endogenous TRAF6 physically interacts with JARID1B in PC3 cells, as shown by reciprocal co-immunoprecipitation between the two proteins (Also see Supplementary Figure S5). (D) In vivo ubiquitination assay shows that TRAF6 increases K63-linked ubiquitination of JARID1B and SKP2 inhibits TRAF6-mediated JARID1B ubiquitination. Cells were transfected with Flag-JARID1B, HA-Ub-K63-only, Myc-TRAF6 and Myc-SKP2 constructs as indicated. WCL indicates the whole cell lysates. (E) TRAF6 mediates JARID1B ubiquitination through lysine residue 242. HEK293T cells were transfected with Flag-JARID1B WT or Flag-JARID1B-K242R, HA-Ub-K63-only, Myc-TRAF6 and Myc-SKP2 plasmids as indicated. In vivo ubiquitination assay was performed in a standard procedure. WCL indicates the whole cell lysates.