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. 2016 Apr 22;17(6):842–857. doi: 10.15252/embr.201541402

Figure 1. UBQLN4 associates with newly synthesized polyubiquitinated proteins.

Figure 1

  1. UBQLN4 associates with polyubiquitinated proteasomal substrates. Flag‐tagged UBQLN4 protein was affinity‐purified from extracts of HeLa cells that were treated with (+) or without (−) 20 μM MG‐132 for 4 h before harvesting, and the Flag precipitates (IP:Flag) were blotted with anti‐polyubiquitin (FK2), anti‐BAG6, and anti‐Flag antibodies. Mock indicates empty vector transfection. Note that UBQLN4‐BAG6 co‐precipitation can be observed exclusively in the presence of polyubiquitinated substrates.
  2. Polyubiquitinated proteins associated with UBQLN4 are sensitive to the translation inhibitor cycloheximide (CHX). HeLa cells expressing Flag‐tagged UBQLN4 were treated with 5 μM MG‐132 as well as CHX (either 10 or 25 μg/ml) for 4 h as indicated and then subjected to immunoprecipitation with an anti‐Flag antibody as in (A).
  3. Schematic representation of MG‐132 and CHX chase experiments in (D). During the course of MG‐132 (5 μM) treatment for 4 h, CHX (10 μg/ml) was added at the indicated time points before harvesting.
  4. HeLa cells were treated as in (C), and Flag precipitates were blotted as in (B).

Source data are available online for this figure.