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

Figure 5. Endogenous Usp27x stabilizes Bim in Caco2 and 293FT cells.

Figure 5

  1. Loss of endogenous Usp27x enhances destabilization of BimEL in response to BRAF‐V600E. Caco2 cells carrying inducible HA‐BRAF‐V600E were transfected with control siRNA (siCo3) or siRNA directed against Usp27x (combination of three siRNAs targeting Usp27x mRNA) for 24 h prior to BRAF‐V600E expression with dox for 2 (left) or 4 h (right). BimEL was detected by Western blotting. Similar results were also observed in n = 2 more experiments after 3 h induction (not shown, see Fig EV6E for siRNA efficacy).
  2. 293FT cells deficient for Usp27x show enhanced destabilization of BimEL in response to PMA. 293FT wt cells or 293FT‐Usp27xKO (clone 2/10, generated using CRISPR/Cas9) were treated with PMA (16.2 nM) for 16 or 24 h to induce BimEL degradation. Western blots show protein levels of n = 3 independently performed experiments (similar results were obtained in one more experiment, not shown). See Fig EV6G for verification of the Usp27xKO cell line by DNA sequence analysis.