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. 2010 Aug 3;285(41):31139–31147. doi: 10.1074/jbc.M109.078808

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — LSF SP/TP motifs and the WW binding domain of Pin1 are required for association of LSF with Pin1. A, NIH 3T3 cells were transfected with p3X FLAG-Pin1 (WT) and cotransfected with either the parental pCMV-QZ construct (−), pCMV-LSF (WT), or pCMV expressing LSF 8AP, a mutant LSF in which all eight Ser-Pro and Thr-Pro motifs are replaced by Ala-Pro dipeptides. FLAG-Pin1 was immunoprecipitated from cellular extracts with anti-FLAG-M2 antibody and both the immunoprecipitates and input extracts were analyzed by immunoblotting with anti-LSF-314. Results are representative of six independent experiments. B, NIH 3T3 cells were cotransfected with expression constructs for wild-type LSF and either p3X FLAG-Pin1 (WT), a WW domain mutant of Pin1 (Y23A), or a catalytic domain mutant of Pin1 (R68A/R69A). Extracts were immunoprecipitated with anti-FLAG-M2 antibody, and coimmunoprecipitated proteins and input extracts were analyzed as in B. Input extracts were also analyzed for exogenous Pin1 levels by immunoblotting with anti-FLAG-M2 antibody. Results are representative of two independent experiments.