Ubiquitination profile is altered in the dTAK1^K142R mutant.

Abstract

<p>(A) Mutant dTAK1^K142R showed enhanced ubiquitination. Expression vectors encoding dTAK1-V5 or dTAK1^K142R-V5 were transfected in S2 cells along with dTAB2-HA and cMyc-Ub in combinations as shown. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc. Observe the enhanced degradation of dTAK1^K142R-V5 in the cell lysate (right lane). (B) Mutant dTAK1^K142R showed enhanced K48-linked ubiquitination and very little K63-linked ubiquitination. Expression vectors encoding dTAK1-V5 or dTAK1^K142R-V5 were transfected in S2 cells along with dTAB2-HA and cMyc-UbK63 (left panel) or cMyc-UbK48 (right panel) in combinations as shown. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc antibody. Protein size markers (NEB) are depicted adjacent to the top panels with values given in kDa.</p