Figure 3
UBR5-containing EDVP E3 ligase complex interacts and regulates MOAP-1 ubiquitylation and stability. (a) Flag-MOAP-1 was transfected into 293T cells, and lysates were prepared 48 h posttransfection. Co-IP with Flag M2 agarose beads were performed and immunoblotted with antibodies as indicated. n=3 independent experiments. (b) In vitro ubiquitylation assay was performed as Figure 2d in the presence or absence of recombinant DDB1, VprBP and Dyrk2. n=3 independent experiments. Original uncropped image is shown in Supplementary Figure S6. (c) siCtrl or siDyrk2 was transfected into H1299 or 293T cells, and Flag-MOAP-1 was transfected into each siRNA transfectant 24 h post-siRNA transfection. Cell lysates were prepared under denaturing condition; Flag-MOAP-1 was immunoprecipitated and immunoblotted with ubiquitin antibody. Same membrane was re-blotted with Flag antibody for Flag-MOAP-1. n=3 independent experiments. Asterisk in Dyrk2 blot indicates non-specific band. Quantification of MOAP-1 ubiquitylation is shown in Supplementary Figure S3B. (d) 293T cells transfected with siCtrl, siDyrk2 or siUBR5 were treated with CHX 48 h posttransfection. Cells were collected at the indicated times after CHX treatment, and lysates were prepared and immunoblotted as indicated (left). MOAP-1 protein level was quantified and plotted (right). The MOAP-1 abundance at 0 time point was set at 100%. n=4 independent experiments (means±s.e.m.). Molecular weight markers are in kDa.
