Newly synthesized defective polypeptides are endogenous clients of TanGIBLE. (A) TanGIBLE retrieves heat shock–induced polyubiquitinated proteins. HeLa cells were co-transfected with Flag-tagged TanGIBLE and T7-tagged ubiquitin, and these cells were cultured at 44°C (heat stress, +) or 37°C (without heat stress, −) for 1 h without the proteasome inhibitor. After heat shock treatment, Flag-tagged probes were immunoprecipitated (IP) and blotted with anti-T7 antibody. Mock indicates empty vector transfection. (B) T7-tagged TanGIBLE can co-precipitate endogenous polyubiquitinated proteins after heat stress. TanGIBLE and its control probes were immunoprecipitated from heat-stressed cells using anti-T7 agarose beads, and co-precipitates of endogenous polyubiquitinated proteins were detected using anti-polyubiquitin FK2 antibody. α-Tubulin was used as the loading control. (C) HeLa cells expressing Flag-tagged TanGIBLE and its 10S negative control fragment were treated with 10 μM MG-132. Flag-precipitates were blotted using anti-polyubiquitin FK2 antibody (upper panel). (D) Polyubiquitinated proteins associated with TanGIBLE are sensitive to CHX. HeLa cells expressing Flag-tagged TanGIBLE or TR-TUBE were treated with 25 μg/ml CHX (or its solvent, EtOH) and then 10 μM MG-132 as indicated for 4 h, then were affinity-purified from the cell extracts using an anti-Flag antibody. Flag-precipitates (IP: Flag) were blotted with anti-polyubiquitin FK2 antibody to detect co-precipitated endogenous polyubiquitinated proteins. Source data are available for this figure: SourceData F4.