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. 2024 Jan 30;115(4):1196–1208. doi: 10.1111/cas.16090

FIGURE 5.

FIGURE 5

Thiolutin, a BRCC36 inhibitor, affects FLT3‐mediated signaling and K63 polyubiquitin. (A) 293T cells were transfected with HA‐K63‐Ub and ITD or TKD. The expression levels of FLT3 and HA‐K63‐Ub were detected by western blot using anti‐FLT3 and anti‐HA antibodies. After treatment with thiolutin (Thl), the expression levels of p‐STAT5 in Ba/F3 cells (B) and the expression levels of p‐FLT3, FLT3, and p‐STAT5 in 293T cells (C) were detected by western blot. The relative expression levels of p‐STAT5 were calculated by the intensities of p‐STAT5 to that of STAT5, which for cells without treatment with Thl was set as 1.0. Data were analyzed by one‐way ANOVA and presented as the mean ± SD (n = 3). α‐Tubulin was used as an internal control. (D) After treatment with Thl, the expression levels of p‐FLT3, FLT3, and p‐STAT5 in RS4‐11 and MV4‐11 cells were detected by western blot. The relative expression levels of p‐STAT5 were calculated by the intensities of p‐STAT5 to that of STAT5, which for MV4‐11 cells without treatment with Thl was set as 1.0. (E) 293T cells expressing HA‐K63‐Ub and FLT3‐ITD or TKD were treated with or without thiolutin (left panel), BRCC36‐siRNA (middle panel), or BRCC36‐overexpression plasmid (right panel). After immunoprecipitation with an anti‐FLT3 antibody, the expression levels of K63 polyubiquitin in the immunoprecipitants were detected by western blotting with an anti‐HA antibody. (F) The diagrams show the WT and mutants used in this study. The gray shading shows the REYEYDL(K) amino acid sequence, duplicated in the ITD. TM, transmembrane domain; JM, juxtamembrane domain. (G) The effects of lysine at 609 of ITD on K63 polyubiquitin and interaction between ITD and BRCC36. The immunoprecipitants of FLT3 were detected by western blotting with indicated antibodies. The relative expression levels of BRCC36 were calculated by the intensities of BRCC36 against FLT3, which for ITD cells was set as 1.0. (H) The expression levels of FLT3 in the same amounts of cell lysates were evaluated by western blot. α‐Tubulin was used as an internal control. *P < 0.05, **P < 0.01, ***P < 0.001.