Figure 2 | Scientific Reports

Figure 2

From: The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL

Figure 2

SH2-U-box interacts with BCR-ABL or BCR-ABL T315I and promotes their ubiquitination and degradation.

(A) 293 T cells were transfected with the indicated constructs and the interaction between SH2-U-box and BCR-ABL or BCR-ABL T315I were determined by co-immunoprecipitation assay. (B) 293T cells were transfected with the indicated constructs together with pcDNA3.1(+)-3 × HA-Ub and treated with MG-132 (10 μM) for 4–6 hours. BCR-ABL/BCR-ABL T315I ubiquitination were assessed by in vivo ubiquitination assay as described in Methods. Whole cell lysates (WCL) were subjected to Western blotting with an anti-FLAG. (C) 293T cells transfected as indicated were treated with CHX (50 μg/ml) for 0, 4, 6 and 12 h in the absence or presence of MG132 (10 μM). BCR-ABL/BCR-ABL T315I protein stability was analyzed by Western blotting and quantified as relative band intensity from three independent experiments (n = 3).

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