Involvement of Posttranslational Modification of NCAPP1 for Binding to Phloem Proteins.(A) Phosphorylation and glycosylation status of in planta–expressed.

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Involvement of Posttranslational Modification of NCAPP1 for Binding to Phloem Proteins.(A) Phosphorylation and glycosylation status of in planta–expressed and purified recombinant Nt-NCAPP1. Involvement of Posttranslational Modification of NCAPP1 for Binding to Phloem Proteins.(A) Phosphorylation and glycosylation status of in planta–expressed and purified recombinant Nt-NCAPP1. Protein gel blot analyses were performed with anti-Nt-NCAPP1 polyclonal antibodies and antiphosphoserine, antiphosphotheronine, and anti-O-GlcNAc monoclonal antibodies. CIP and GDase pretreatment of purified recombinant Nt-NCAPP1 confirmed the specificity of these immunoreactions. Lane 1, partially purified GSH; lane 2, partially purified Nt-NCAPP1ΔN-GSH.(B) FPLC-fractionated pumpkin phloem proteins stained with SYPRO Ruby.(C) FPLC-fractionated phloem proteins were blotted onto membrane, overlaid with recombinant Nt-NCAPP1ΔN-GSH, and Nt-NCAPP1 interaction partners detected by anti-NCAPP1 polyclonal antibodies.(D) GSH control. FPLC-fractionated phloem proteins were blotted onto membrane, overlaid with GSH, and then probed with anti-NCAPP1 polyclonal antibodies.(E) FPLC-fractionated phloem proteins were blotted onto membrane, overlaid with E. coli–expressed recombinant His-tagged Nt-NCAPP1ΔN (R-His-Nt-NCAPP1ΔN), and Nt-NCAPP1 interaction partners detected by anti-NCAPP1 polyclonal antibodies.(F) BSA control. FPLC-fractionated phloem proteins were blotted onto membrane, overlaid with BSA, and then probed with anti-NCAPP1 polyclonal antibodies.(G) to (I) Nt-NCAPP1ΔN-GSH overlay assays performed on FPLC-fractionated phloem proteins. Purified recombinant Nt-NCAPP1ΔN-GSH was pretreated with CIP, GDase, or both CIP and GDase, respectively; interaction proteins were detected by anti-Nt-NCAPP1 polyclonal antibodies. Note that phosphorylation and glycosylation on Nt-NCAPP1 is necessary for binding to Cm-PP16-1 and other phloem proteins.Boxed areas indicate the location of native Cm-PP16-1 (top band) and Cm-PP16-2 (bottom band). Ken-ichiro Taoka et al. Plant Cell 2007;19:1866-1884 ©2007 by American Society of Plant Biologists