Figure 6.

PDGLP1 Interacts with a Subset of Proteins in the PECP Fraction to Form a PDGLP1-Specific Complex.

(A) Protein overlay assays establish that PDGLP1-MHT interacts with a range of proteins contained in the BY-2 cell PECP preparation. Proteins in the PECP were separated by fast protein liquid chromatography, and fractions were then separated by SDS-PAGE, followed by transfer to nitrocellulose membrane. Membrane was overlaid with purified PDGLP1-MHT and then subjected to immunoblot analysis, using an anti-c-Myc monoclonal antibody, to identify interacting proteins. GFP-MHT was used as a negative control.

(B) PDGLP1-interacting proteins identified by co-IP experiments. Purified recombinant GFP, PDGLP1-GFP (lane 1), or PDGLP1-Myc (lane 2) was incubated with an Arabidopsis PECP fraction (Input) followed by co-IP using anti-GFP or anti-c-Myc monoclonal antibodies (mAb). White, black, and red asterisks indicate immunoprecipitated GFP, PDGLP1-GFP, and PDGLP1-Myc, respectively. GFP served as the negative control. Immunoprecipitated bait proteins were confirmed by immunoblot analysis using anti-GFP (middle panel) or anti-c-Myc mAb (right panel).

(C) Both PDGLP1-GFP and PDGLP1-Myc are highly enriched in the Arabidopsis PECP fraction. Transgenic P_PDGLP1:PDGLP1-GFP or P_35S:PDGLP1-Myc plants were used to prepare soluble and PECP fractions, which were then separated by SDS-PAGE and tested by protein gel blot analysis using GFP or anti-c-Myc monoclonal antibodies. PECP fractions were prepared from leaf (LF) and root (RT) tissues. S, soluble fraction; P, PECP fraction.

[See online article for color version of this figure.]