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. 2016 Mar 24;17(5):724–738. doi: 10.15252/embr.201541392

Figure 6. Usp27x stabilizes Bim protein levels in the ERK‐degradation pathway in 293FT cells, 1205Lu melanoma and HCC827 NSCLC cells.

Figure 6

  1. 293FT‐TetR‐3xFlagUsp27x cells were treated with PMA (16.2 nM) plus QVD (10 μM) and 3xFlag‐Usp27x was induced by dox at the same time for 24 h followed by addition of cycloheximide (CHX). Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. Similar results were obtained in n = 3 separate experiments. Cut membranes shown were from one membrane with same exposure time. For each condition, BimEL levels were quantified and normalized to the tubulin signal. Per cent BimEL gives the expression relative to the starting point.
  2. 1205Lu melanoma cells carrying dox‐inducible GFP‐Usp27x were treated with dox for 24 h. Cycloheximide (CHX, 1 μg/ml) was then added. Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. GFP‐Usp27x was detected with anti‐GFP antibodies. Similar results were obtained in n = 3 separate experiments. A second experiment is shown in the right panel also including 1205Lu melanoma carrying dox‐inducible GFP‐Usp27xC87A mutant. For each condition, BimEL levels were quantified and normalized to the tubulin signal. Per cent BimEL gives the expression relative to the starting point set to 100%. The starting point for the non‐induced Usp27x/C87A cells (‐dox) is shown in lane 1 for experiment one and two (‐dox, ‐CHX). Cut membranes shown were from one membrane with same exposure time. Same results were obtained for WM1158 melanoma cells (data not shown).
  3. HCC827 cells were induced to express GFP‐Usp27x with dox as indicated. Twenty‐four hours later, cycloheximide (CHX, 1 μg/ml) was added for the indicated time. BimEL was detected after the indicated times of treatment by Western blotting. For each condition, BimEL levels were quantified and normalized to the tubulin signal. Per cent BimEL gives the expression relative to the starting point. Similar results were obtained in n = 2 separate experiments. Cut membranes shown were from one membrane with same exposure time.