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

Figure EV6. Usp27x stabilizes Bim.

Figure EV6

  1. Overexpression of Usp27x but not Usp22 stabilized endogenous BimEL in 1205Lu melanoma cells. 1205Lu cells carrying inducible constructs were treated with dox to induce GFP‐Usp27x, GFP‐Usp27xC87A or 3xFlag‐tagged Usp22 for 48 h. Bim was detected by Western blotting. Similar results were obtained in n = 2 separate experiments.
  2. 1205Lu cells with inducible constructs were treated as indicated to induce GFP or GFP‐Usp27x. The MEK inhibitor UO126 was used to block ERK activity. Of note, BimEL protein shifted to a lower molecular size when cells were treated with UO126, presumably as an effect of dephosphorylation. Similar results were obtained in n = 2 separate experiments.
  3. Caco2 colon carcinoma cells carrying dox‐inducible HA‐BRAF‐V600E were treated as indicated. ERK phosphorylation was measured by Western blotting. Note the loss of Bim protein upon induction of HA‐BRAF‐V600E (n = 2).
  4. 3xFlagUsp27x inhibits Bim loss upon BRAF‐V600E induction. Maternal Caco2‐HA‐BRAF V600E cells or the same cells stably expressing constitutive 3xFlag‐Usp27x were treated with dox for 3 (left) or 96 h (right) to induce BRAF‐V600E (two separate experiments). Bim levels were measured by Western blotting. For each condition, BimEL levels were quantified from the shown immunoblot and normalized to the tubulin signal. Per cent BimEL gives the expression relative to the starting point set to 100%. Similar results were obtained in n = 3 separate experiments with varying induction times. See also Fig 4D.
  5. Validation of Usp27x‐specific siRNAs. 293FT cells were transfected with siRNAs targeting human Usp27x or with control siRNA. Knockdown of Usp27x was either tested by using one single siRNA against Usp27x (#861) or by using a combination of three siRNAs targeting Usp27x (#495, #498, #861). After 24 h, cells were transfected with a construct driving expression of human Usp27x and expression of GFP off an IRES. Usp27x and GFP were detected 24 h later by Western blotting (n = 2). The combination of three siRNAs was used for the later knockdown experiments (see Fig 5A).
  6. Comparison of Bim expression levels between 293FT and 1205Lu. Western blot of whole‐cell lysates obtained from 293FT cells and from 1205Lu melanoma cells. For both cell lines, 50 μg protein was run by SDS–PAGE, and Bim was identified using antibody against Bim. Tubulin served as a loading control. Similar results were obtained in n = 2 separate experiments.
  7. Validation of 293FT‐Usp27xKO (clone 2/10). Sequencing results (n = 1) indicate a thymidine (T) insertion a nucleotide (nt) 391 (arrow). This insertion leads to the addition of 14 different amino acids after aa 130 followed by a stop codon. Therefore, this clone expresses a truncated version of Usp27x (wt has 438 aa) deleting part of the catalytic activity/triad (involves aa C87A (nucleophile), and the predicted sites H380 (proton acceptor) and aa D396/D397 in Usp27x 28).