Figure 4. UBA domain of UBQLN1 is responsible for stabilization of BCLb^WT.

293T cells were transfected with MIG-BCLb^WT, MIG-BCLb^WT, MIG-BCLb^ΔTM, FLAG-UBQLN1^WT, domain deletion constructs FLAG-UBQLN1^112X and FLAG-UBQLN1^ΔUBA as indicated. 36 hours post transfection, cells were treated with the indicated inhibitor for 16 hours; vehicle (−), translational elongation inhibitor Cycloheximide (CH) and proteasomal inhibitors MG132 (MG), Epoximicin (Epoxi.) and Bortezomib (Bortez.). Cells were lysed and Western Blot analyses were performed with the indicated antibody. (A) Stability of BCLb^WT and BCLb^K0 was tested in the presence of UBQLN1, when exposed to the indicated inhibitors for 16 hours. (B) Experiment was performed as in A but in the presence of UBQLN1. As seen in A, expression of BCLb^WT is lost upon exposure to CH for 16 hours, but in the presence of UBQLN1, it is stabilized. Loss of BCLb^WT seen with proteasomal inhibitors is also rescued by the presence of UBQLN1. UBQLN1 also prevents loss of BCLb^K0 seen with MG132. (C) Experiment was performed as in A in the presence of CH and domain deletion constructs of UBQLN1 and we mapped the UBA domain to be responsible for stabilizing BCLb^WT. Both UBQLN^112X and UBQLN1^ΔUBA failed to prevent loss of BCLb^WT. (D) Stability of BCLb^ΔTM was tested similar to BCLb^WT and BCLb^K0. BCLb^ΔTM is generally less stable than BCLb^WT and presence of UBQLN1 does not alter its expression.