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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 3. — ERK8 interacts with PCNA via a PIP box. (A and B) Chromatin was immunoprecipitated with antibodies against ectopically expressed HA-tagged ERK8 (A) or endogenous PCNA (B), treated with DNase I, and analyzed by immunoblotting. A fraction of the input is shown for comparison to the level of associated PCNA or ERK8. (C) GST–ERK8 (PIP) fusion proteins were immunoprecipitated with antibodies to PCNA. The soluble fraction of MCF-10A lysates was incubated with GST-PIP box fusions containing the wild-type (QALQHPYVQRFH) or mutant ERK8 PIP (QALAHPYVQRFH) box. PCNA was immunoprecipitated and electrophoresized, and associating proteins were analyzed by immunoblotting. A fraction of the input is shown for comparison to the level of associated GST–ERK8 (PIP). (D) Chromatin was immunoprecipitated with antibodies (Ab) against ectopically expressed HA-tagged wild type or ERK8(Q300A) mutant, treated with DNase I, and analyzed by immunoblotting. A fraction of the input is shown for comparison to the level of associated PCNA. I, insoluble fraction; IP, immunoprecipitation; S, soluble fraction.