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. 2016 Nov 29;8(2):3144–3155. doi: 10.18632/oncotarget.13673

Figure 5. RNF2 is a E3 ligase of SIK1 and ubiquitinates SIK1.

Figure 5

A and B. RNF2 interacts with SIK1 in vivo. HCC cell lysates were subject to immunoprecipitation with anti-IgG, anti-SIK1 (A), or anti-RNF2 (B) antibodies. The immunoprecipitates were subsequently blotted with the indicated antibodies. C. RNF2 interacts with SIK1 in vitro. SIK1 was purified from bacteria and incubated with GST or GST–RNF2 coupled to GSH–Sepharose. The proteins retained on the Sepharose beads were subsequently blotted with the indicated antibodies. D. The knockdown of RNF2 increases SIK1 protein levels, but not SIK2 or SIK3(left panel), RNF2 overexpression decreases SIK1 protein expression (right panel). E. HCC cells were transfected with the indicated shRNAs. After 72 h, the proteins were extracted and subjected to Western blot. F and G. RNF2 depletion or overexpression affects SIK1 stability respectively. HCC cells transfected with the indicated constructs were treated with CHX (0.1 mg/mL) and harvested at the indicated time. The cell lysate was subjected to Western blot analysis. H and I. ubiquitination assay of SIK1. Immunoblots of SIK1 and RNF2.