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. 2012 Oct 31;287(52):43639–43650. doi: 10.1074/jbc.M112.393678

FIGURE 5.

FIGURE 5.

CG-5 facilitated β-TrCP1-mediated HuR ubiquitination in LNCaP cells. A, coimmunoprecipitation analysis of the time-dependent effect of CG-5 (5 μm) on HuR ubiquitination. LNCaP cells ectopically expressing HA-Ubiquitin (HA-Ub) were treated with 5 μm CG-5 for 12 or 36 h, followed by cotreatment with proteasome inhibitor MG132 for an additional 12 h. Equal amounts of cell lysates were immunoprecipitated (IP) with anti-HuR antibody and protein A/G-agarose followed by immunoblotting (WB) with anti-HA antibodies. B, ectopic expression of ΔF-β-TrCP1-Myc blocked CG-5-mediated HuR ubiquitination. LNCaP cells cotransfected with plasmids encoding HA-Ub, HuR-FLAG, and ΔF-β-TrCP1-Myc or empty vector (pCMV) were treated with 5 μm CG-5 for 12 or 36 h, followed by cotreatment with proteasome inhibitor MG132 for an additional 12 h. Equal amounts of cell lysates were immunoprecipitated with anti-FLAG antibody and protein A/G-agarose followed by immunoblotting with anti-HA and anti-FLAG antibodies.