Figure 5

TGF-β upregulates USP15 translation. (a) TGF-β promotes the USP15 synthesis in U2OS cells. U2OS cells were cultured in the absence or presence of TGF-β for 16 h. Cell lysates were prepared and probed for USP15, p53, p21 and actin. (b) Quantification of a. The results are shown as a percentage of the protein/actin ratios without TGF-β treatment and are averaged from three independent experiments (means±s.d.). (c) USP15 knockdown abolishes p53 stability and p53 transcriptional activation mediated by TGF-β. U2OS cells with USP15 knockdown were cultured in the absence or presence of TGF-β for 24 h. Cell lysates were prepared and probed for USP15, p53, p21 and actin. (d) TGF-β does not affect USP15 transcription. USP15 mRNA of HEK293 cells was quantitated by real-time PCR as described in 'Materials and methods' section and normalized to GAPDH mRNA. (e) TGF-β enhances USP15 translation. HEK293 cells pre-treated with MG132 (20 μM) for 5 h were cultured for 16 h in the absence or presence of TGF-β (2 ng/ml). (f) TGF-β does not affect USP15 degradation. HEK293 cells pre-treated with cycloheximide (100 μg/ml) for 5 h were treated with or without TGF-β (2 ng/ml) for 16 h. Cell lysates were analyzed by western blotting and probed for USP15 and actin.