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ERK5 activation induces p53 ubiquitination via increasing CHIP ubiquitin ligase activity. (A) After transduction with Ad-CA-MEK5α or the Ad-LacZ control, HUVECs were exposed to 15d-PGJ2 for 4 h. The level of p53 mRNA was determined by using real time quantitative RT-PCR as described in the Materials and Methods section. (B) The endogenous level of p53 ubiquitination was determined by immunoprecipitation analysis with anti-p53 antibody, followed by immunoblotting with an anti-ubiquitin antibody. (C) CHIP ubiquitin ligase activity was determined by an in vitro ubiquitination assay kit (BostonBiochem) with a GST-fused p53 recombinant protein as a substrate. HUVECs were transduced with Ad-CA-MEK5α and then followed by treatment with 10 µM BIX02189. Cell lysates were applied for immunoprecipitation analysis with an anti-CHIP antibody. Immunoprecipitated CHIP was incubated with recombinant proteins including ubiquitin and E1/E2 enzyme mixture, for 60 min at 37. CHIP ubiquitin ligase activity was determined by immunoblotting with an anti-ubiquitin antibody. The protein amount of GST-p53 and immunoprecipitated CHIP was detected by Western blot analysis with anti-p53 and anti-CHIP antibodies. Data are representative of three independent experiments. ERK5, extracellular signal-regulated kinase 5; CHIP, C terminus of Hsc70-interacting protein; Ad-CA-MEK5α, adenovirus encoding constitutively active form of MEK5 alpha; 15d-PGJ2, 15-deoxy-Δ(12,14)-prostaglandin J2; RT-PCR, reverse transcriptase-polymeratse chain reaction; HUVEC, human umbilical vein endothelial cells; IP, immunoprecipitation; IB, immunoblot; NS, non-significant.