Outline of phage library screening.

<p>A sequential strategy was used for screening of complete as well as subsequent rationally customized SH3 display libraries in order to exhaustively assess potential SH3 partners of the indicated ADAMs.</p

Cytosolic tails of ADAM-metalloproteases and their candidate SH3 binding motifs.

<p><b>A.</b> Schematic presentation of ADAM cytosolic tails with the location of the candidate SH3-binding proline clusters indicated by circles with roman numerals counting from the transmembrane region to the carboxy terminus. The scale bar indicates distance in amino acid residues. <b>B.</b> Potential SH3 binding sequences within the proline clusters shown in A. Established SH3 target motifs (+ΦPxxP, PxΦPx+, PxxDY, where + is K or R and Φ is a hydrophobic residue) occurring individually or in clusters where they partly overlap each other are indicated in bold. <b>C.</b> Western blotting analysis of the ADAM tails expressed as biotinylated fusion proteins in human 293T cells for use as affinity baits in SH3 domain library screening.</p

Summary of the peptide array data for identification of functional SH3 binding sites in the ADAM tails.

<p>The indicated peptides corresponding to the proline-cluster numbering in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121301#pone.0121301.g001" target="_blank">Fig 1A</a> were printed in triplicate on arrays slides, and probed with the indicated SH3 domains. Signals of the triplicate spots were averaged, and these values normalized to the average of the peptide giving the strongest signal in each SH3 probing. Shown is a heat map based on these normalized values where white indicates background level binding, and black the strongest peptide binding for each SH3 domain.</p

Key statistics of phage library selection experiments.

<p>Key statistics of phage library selection experiments.</p

ADAM8 interacts with TOCA1 and SNX33 in human cells.

<p>SNX33 and TOCA1 were expressed as EGFP-fusion proteins by transiently transfected 293T cells alone or with HA-tagged ADAM8 as indicated on top of the figure. The presence of ADAM8 itself (top panel) or TOCA1 or SNX33 that was associated with it (second panel from top) in anti-HA immunoprecipitates (IP) from the lysates of these cells was examined by Western blotting. Similar total levels of ADAM8, TOCA1, and SNX33 between the transfected cells were confirmed by a Western blotting analysis of the unselected lysates (Lysate). Blotting for the endogenous α-tubulin in these lysates was included as an additional loading control.</p