Figure 2. The transmembrane domain of BCLb is recognized by STI-1/2 domains of UBQLN1 and stabilizes BCLb in the cytoplasm.

293T cells were transfected with empty vector (EV), FLAG-UBQLN1^WT, or FLAG-tagged domain deletion constructs, MYC-BCLb^WT or MYC-BCLb^ΔTM. 48 hours post transfection, cells were lysed and Western Blot analyses were performed with the indicated antibody. Where indicated, immunoprecipitation with anti-FLAG conjugated agarose beads was performed to determine interaction between UBQLN1 and BCLb proteins. (A) STI-1/2 domains of UBQLN1 are required to interact with BCLb. (B) BCLb^ΔTM does not interact with UBQLN1. A functional transmembrane domain on BCLb is required for UBQLN1 to recognize and interact with it. (C) 293T cells expressing BCLb^WT or BCLb^ΔTM with EV or FLAG-UBQLN1, were subjected to subcellular fractionation, followed by Western Blot analyses. Cells were fractionated into cytosolic (c) and membranes (m). VDAC1, a membrane bound protein, and Tubulin, a cytoplasmic protein were used to document the validity of the fractionations. UBQLN1 does not interact with BCLb^ΔTM and therefore does not alter the location of BCLb^ΔTM, as it is unable to recognize it.