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Carboxyl terminus of palladin interacts with MRTFs in SMCs.

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posted on 2010-09-22, 02:40 authored by Li Jin, Qiong Gan, Bartosz J. Zieba, Silvia M. Goicoechea, Gary K. Owens, Carol A. Otey, Avril V. Somlyo

A) Full length, amino (N), or carboxyl (C) terminus of myc tagged palladin constructs were cotransfected with flag tagged MRTF-A or B into HEK-293 cells. Co-IP assays were performed, and proteins were detected with myc and flag antibodies. Note that different regions of the gels are shown for full length, and the N- and C-termini, which as expected, run differently, as shown in Fig. 2C. B) in vitro pull down assays showed that MRTF-A interacts with palladin directly. Recombinant GST tagged palladin was purified from E.Coli and MRTF-A and –B proteins were translated in vitro. C) MRTF-A interaction with palladin is not via the amino terminal RPEL domain. The two RPEL motifs (mut1: R33A and P34A and mut2: L39A and V40A) mutants and an amino terminal 100 aa deletion of MRTF-A (ΔN) were cotransfected with myc tagged palladin into HEK-293 cells, and co-IP was performed 36 h after transfection with anti-myc antibody and blotted with anti-myc or flag antibodies.