Physical association of AP2, TPL and HDA19.

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Physical association of AP2, TPL and HDA19. Physical association of AP2, TPL and HDA19. (A) Yeast two-hybrid (Y2H) assays. Protein domains of TPL and AP2 are depicted. TPL and AP2 interact by Y2H (bottom right) as indicated by blue coloration. Deletion of the CTLH domain or introduction of the N176H missense mutation into the TOP domain of TPL disrupts interaction with AP2. Mutation of the conserved leucines within the EAR motif of AP2 prevents binding to TPL. (B) In a semi-in vivo pull-down assay, GST N-TPL binds AP2-GFP from floral bud lysates but not AP2 mEAR-GFP. GST N-TPL N176H does not bind AP2-GFP. Both GST N-TPL and GST N-TPL N176H interact with HDA19-HA. Ponceau Red and Coomassie Blue staining shows equal protein loading and efficient GST protein expression, respectively. (C) Bimolecular fluorescence complementation (BiFC) assays. Nuclear fluorescence (arrowheads) shows physical association between HDA19 and both TPL and TPL N176H in transiently transfected tobacco cells. Homodimerization of TPL serves as a positive control; the right column depicts negative control combinations. Naden T. Krogan et al. Development 2012;139:4180-4190 © 2012.