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. 2017 Mar 31;36(10):1434–1446. doi: 10.15252/embj.201695669

Figure 2. USP49 deubiquitinates and stabilizes FKBP51.

Figure 2

  1. SU86 cells stably expressing control or USP49 shRNAs were treated with vehicle or MG132. Half of the cells are lysed, and Western blot was performed with the indicated antibodies. Lower panel: The mRNAs were extracted from the rest of the cells and subjected to qRT–PCR. Error bars represent the SEM of three independent experiments.
  2. SU86 cells stably expressing control (Ctrl) or USP49 shRNAs were transfected with the indicated constructs. After 48 h, half of the cells were lysed and Western blot was performed with the indicated antibodies. Lower panel: The mRNAs were extracted from the rest of the cells and subjected to qRT–PCR. Error bars represent the SEM of three independent experiments.
  3. SU86 cells stably expressing control (Ctrl) or USP49 shRNAs were treated with CHX (0.1 mg/ml) and harvested at the indicated times. Cells were lysed and cell lysates were then blotted with the indicated antibodies. Lower panel: quantification of the FKBP51 protein levels relative to β‐actin. Error bars represent the SEM of three independent experiments.
  4. Cells transfected with the indicated constructs were treated with or without MG132 for 4 h before harvest. FKBP51 was immunoprecipitated and immunoblotted with the indicated antibodies.
  5. Cells stably expressing control (Ctrl) or USP49 shRNAs were treated with MG132 for 4 h before harvest. FKBP51 was immunoprecipitated and immunoblotted with the indicated antibodies.
  6. Deubiquitination of FKBP51 in vitro by USP49. Ubiquitinated FKBP51 was incubated with purified USP49 or USP49CA in vitro and then blotted with the indicated antibodies.