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

Figure 4. Usp27x but not Usp22 can counter Bim destabilization through the Raf‐ERK pathway.

Figure 4

  1. Lines based on the human melanoma cell line 1205Lu (BRAF‐V600E‐positive) were made to carry GFP‐Usp27x, GFP‐Usp27xC87A or GFP‐Usp22 under the control of a doxycycline (dox)‐inducible promoter. GFP‐Usp27x, GFP‐Usp27xC87A or GFP‐Usp22 was induced with dox for 24 or 48 h. Levels of Bim were determined by Western blotting. The two left panels are from the same membrane and exposure times. Similar results were obtained in n = 4 separate experiments (left) and n = 3 experiments (right). See also Fig EV6A where 3xFlag‐Usp22 is also shown to be unable to increase Bim levels. GFP‐Usp27x, GFP‐Usp27xC87A or GFP‐Usp22 expression was detected using an antibody against GFP.
  2. A cell line derived from the BRAF‐V600E‐positive human melanoma line WM1158 was generated to carry inducible GFP‐Usp27x. GFP‐Usp27x was induced, and Bim was detected as in (A). Similar results were obtained in n = 3 separate experiments. GFP‐Usp27x expression was detected using an antibody against GFP.
  3. Derivatives of the NSCLC cell line HCC827 (expressing constitutively active EGFR) were generated that only express the Tet repressor (TetR) or carrying in addition either GFP‐Usp27x or GFP‐Usp27xC87A. Cells were treated with dox for 72 h. Bim was detected by Western blotting. Similar results were obtained in n = 2 separate experiments. GFP‐Usp27x or GFP‐Usp27xC87A expression was detected using an antibody against GFP.
  4. Caco2 cells carrying a dox‐inducible HA‐BRAF‐V600E 37 (left two lanes) or the same cells stably expressing 3xFlag‐Usp27x were treated with dox for 48 h to induce expression of HA‐BRAF‐V600E. Bim was detected by Western blotting. The amount of BimEL was quantified from the shown immunoblot using the expression levels of the uninduced control cells set to 100% (normalized to the tubulin signal for each condition). Similar results were obtained in n = 3 separate experiments with varying induction times. See also Fig EV6D.