Figure 1.

The Nbeal2 interactome. (A) Diagram showing the functional domains of Nbeal2 (ARM, Armadillo; ConA, Concanavalin A; WD40 repeat [WD40]). Construct 1 (PBW-FTAP) encompasses the PH, BEACH, and aminoterminal WD40 domains and the FTAP tag. Construct 2 (GFP-FTAP) encompasses GFP and FTAP. (B) Ectopic expression of GFP-FTAP and PBW-FTAP in HEKs. (C) FTAP-purification of GFP-FTAP and PBW-FTAP from HEKs. Purified proteins were resolved by SDS-PAGE, visualized by Coomassie blue stain, and analyzed by liquid chromatography-mass spectrometry. Some lanes in gels from panels B and C were deleted and replaced by black vertical lines. (D) Identified proteins (n = 129) were classified according to their protein class using gene ontology terms. (E) PPIN; 637 bait proteins representing the processes of hemostasis, megakaryopoiesis, and platelet formation were used to retrieve 5112 nodes (proteins) and 26 702 edges (biochemical reactions) by Reactome and IntAct. Orange nodes (n = 91) are interactors present in the PPIN, and the purple nodes (n = 38) have interactions with 1 or more proteins of the PPIN. Nbeal2 is shown in black and Dock7, Sec16a, and Vac14 are shown as orange triangles. (F-H) Subnetworks extracted from the PPIN in panel E for Dock7 (F), Sec16a (G), and Vac14 (H). The expression levels for the corresponding genes determined by the sequencing of RNA of human MKs and expressed as log₂ FPKM (fragments per kilobase of transcript per million mapped reads) is presented in white-to-green color, and only nodes corresponding to genes with FPKM > 1 in MKs are depicted (see color bar for relative FPKM values).⁴³ The gray/black (E-H), light blue (F-H), and dark blue (F-H) edges are based on the results of pull-down experiments reported in this study, Reactome and IntAct, respectively. A Cytoscape file to generate an interactive version of the PPIN, containing gene expression levels and other annotation features, is available from the supplemental data. MW, molecular weight.