Figure 3

Interaction of RNF122 with CAML. (A) In mammalian cells, RNF122 interacted with CAML. HEK293T cells were seeded into 60-mm plates and transfected with pcDB (empty), RNF122-myc or mut-RNF122-myc vectors in conjunction with CAML-FLAG. After 48 h, the cells were treated with 2.5 μg/mL MG132 for 6 h, lysed in PBS containing 1% Triton X-100, and immunoprecipitated with anti-myc antibody agarose beads. (B) CAML is not involved in the proteasome-dependent degradation pathway. HEK293T cells were treated with 2.5 μM MG132 or vehicle (DMSO) for 6 h. The cells were then harvested and the expression of CAML was analyzed by western blotting using an anti-CAML antibody. The membrane was subsequently reprobed with an anti-β-actin antibody. (C) HEK293T cells were transiently cotransfected with CAML-FLAG and pcDB, RNF122-myc, RNF122C92A-myc, or RNF122C95A-myc. At 24 h post-transfection, the cells were treated with 50 μg/mL CHX and harvested at the indicated times. The cell lysates were subjected to SDS-PAGE and analyzed by western blotting by using antisera against FLAG and β-actin. (D) The level of RNF122 was found to increase in a proteasome-independent manner when it was coexpressed with CAML. HEK293T cells were transfected with empty (pcDB) or CAML expression vector along with RNF122-myc, and subsequently treated with 2.5 μg/mL MG132 or DMSO at 24 h post-transfection. After 24 h, the cell lysates were suspended in PBS containing 1% Triton X-100 and a protease inhibitor cocktail, and then immunoblotted with an anti-myc antibody.