Figure 7.

Protein Phosphorylation Is Required for RBP50-Based RNP Complex Formation.

(A) Total phloem sap (containing proteins and mRNA) was treated with or without RNase, followed by co-IP. Purified RBP50-based RNP complexes were then treated with or without CIP (PPase), the proteins were separated by SDS-PAGE, and their profiles were visualized by GBS reagent (left panel). Proteins were then blotted onto nitrocellulose membranes and overlaid with either native phloem-purified RBP50 or BSA, and interacting proteins were detected by anti-RBP50 antibody (Ab; middle and right panels, respectively). Asterisks indicate the bands for CIP.

(B) Total phloem sap was pretreated with RNase and CIP (PPase) before (B) the co-IP with anti-RBP50 antibodies. A second aliquot of total phloem sap was given a CIP treatment after (A) RNase pretreatment and co-IP. Proteins were separated by SDS-PAGE, and their profiles were visualized by GBS reagent (left panel). After blotting onto nitrocellulose membranes, proteins were overlaid with either native phloem-purified RBP50 or BSA, and interacting proteins were detected with anti-RBP50 antibody (middle and right panels, respectively). The asterisk indicates the band for CIP.