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Figure 4 | BMC Cell Biology

Figure 4

From: Multisite phosphorylation of Pin1-associated mitotic phosphoproteins revealed by monoclonal antibodies MPM-2 and CC-3

Figure 4

The direct interaction of Pin1 with CC-3 and MPM-2 mitotic antigens involves its WW domain. (A) Mitotic HeLa cell homogenates were incubated with histidine-binding resins saturated with different his-tagged proteins. WT: wild type Pin1; W34A: a Pin1 protein mutated in the WW domain; C113A: a Pin1 protein mutated in the PPIase domain; Pbp5: an irrelevant protein used as a control. Aliquots from the initial lysate (+), the flowthrough (-), the washes (w1 to w3) and the eluted fractions (E) were subjected to SDS-PAGE and immunoblotting. The Pin1 WT and C113A resins retained more MPM-2 and CC-3 mitotic antigens than the WW mutant W34A and the Pbp5 control resins. (B) Coomassie blue staining of a 15% acrylamide gel of the fractions eluted from the different resins in (A) showing that the initial amounts of the bound his-tagged proteins were similar. (C) Blot overlay experiments using either the wild type or the WW mutant Pin1 protein as a probe showing the direct interaction of wild type Pin1 with CC-3 interphase (lane 3) and mitotic (lane 5) antigens as well as with MPM-2 interphase (lane 4) and mitotic (lane 6) antigens, obtained by immunoprecipitation. Lanes 1 and 2 correspond to total homogenates prepared from asynchronous and mitotic HeLa cells respectively. Pin1 did not bind to the Mr markers (lane M) nor to the major bands of the total homogenates (lanes 1 and 2) visualized by Coomassie blue staining (right panel) but did bind strongly to the CC-3 and MPM-2 antigens. No binding was observed when the W34A mutant protein was used as a probe (middle panel). (D) Pin1 specifically binds to SAP155 and DNA topoisomerase II α. Same experiment as that described in (A) except that the eluted fractions from the his-tagged Pin1 and Pbp5 resins were immunoblotted with antibodies to DNA topoisomerase II α and SAP155.

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