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. 2010 Aug 23;190(4):575–586. doi: 10.1083/jcb.201002124

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© 2010 Groehler and Lannigan

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Figure 5. — ERK8 regulates the association of PCNA with HDM2. (A) Silencing HDM2 enhances PCNA levels in the absence or presence of ERK8. MCF-10A cells were transduced with control (con) or ERK8-specific shRNA, followed by transfection with control or HDM2 siRNA. Lysates were taken 5 d after transduction and divided into insoluble (I) and soluble (S) fractions, normalized for Ran levels, and immunoblotted for PCNA. The top right panel shows the extent of HDM2 knockdown in lysates normalized for Ran levels. Quantitation of the relative amount of PCNA in comparison to Ran and normalized to the appropriate control, which was transduced with luciferase (Luc) shRNA and transfected with control siRNA. Mean is shown (n = 3), and error bars indicate SEM. Silencing HDM2 decreased levels of ubiquitinated PCNA. MCF-10A cells were transduced with ERK8-specific shRNA followed by transfection with a plasmid encoding His6-Ub and control or HDM2 siRNA. PCNA was immunoprecipitated 5 d after transduction, and the normalized PCNA immunoprecipitates were analyzed for ubiquitination. The IgG band is from the immunoprecipitating antibody. (B) Loss of ERK8 increases the association of HDM2 with PCNA. MCF-10A cells were transduced with control or ERK8-specific shRNA for 5 d. PCNA was immunoprecipitated, and the normalized PCNA immunoprecipitates were analyzed for HDM2. The right panel shows the level of HDM2 expression in lysates normalized for Ran. IP, immunoprecipitation; LV, lentivirus; WCE, whole cell extract.