Research Paper Volume 13, Issue 7 pp 10603—10618

Tumor suppressor DCAF15 inhibits epithelial-mesenchymal transition by targeting ZEB1 for proteasomal degradation in hepatocellular carcinoma

Knockdown of CUL4A promotes the stability of ZEB1. (A–D) HEK293T cells were transfected with Flag-CUL4A, Flag-CUL4B, Flag-DDA1 and Flag-DDB1 respectively. After 24h, cells were treated with 10 μM MG132 for 6h. Cell lysates were immunoprecipitated using FLAG-M2 agarose beads and analyzed by immunoblotting with the indicated antibodies. (A) Ectopically expressed CUL4A didn’t bind with endogenous ZEB1. (B) Ectopically expressed CUL4B didn’t bind with endogenous ZEB1. (C) Ectopically expressed DDA1 didn’t bind with endogenous ZEB1. (D) Ectopically expressed DDB1 didn’t bind with endogenous ZEB1. (E) SMMC-7721 cells were transfected with the control or si-CUL1, si-CUL2, si-CUL3, si-CUL4B and si-CUL5 respectively. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Expression of si-CUL1, si-CUL2, si-CUL3, si-CUL4B and si-CUL5 had no effect on ZEB1 degradation, in contrast, si-CUL4A promoted the ZEB1 protein expression.

Figure 4. Knockdown of CUL4A promotes the stability of ZEB1. (AD) HEK293T cells were transfected with Flag-CUL4A, Flag-CUL4B, Flag-DDA1 and Flag-DDB1 respectively. After 24h, cells were treated with 10 μM MG132 for 6h. Cell lysates were immunoprecipitated using FLAG-M2 agarose beads and analyzed by immunoblotting with the indicated antibodies. (A) Ectopically expressed CUL4A didn’t bind with endogenous ZEB1. (B) Ectopically expressed CUL4B didn’t bind with endogenous ZEB1. (C) Ectopically expressed DDA1 didn’t bind with endogenous ZEB1. (D) Ectopically expressed DDB1 didn’t bind with endogenous ZEB1. (E) SMMC-7721 cells were transfected with the control or si-CUL1, si-CUL2, si-CUL3, si-CUL4B and si-CUL5 respectively. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Expression of si-CUL1, si-CUL2, si-CUL3, si-CUL4B and si-CUL5 had no effect on ZEB1 degradation, in contrast, si-CUL4A promoted the ZEB1 protein expression.