PAX-captured proteins are MS identifiable.

<p>(<b>A</b>) PAX captures a known interacting protein following over-expression in FreeStyle 293 cells. An array of peptides consisting of a proline-rich sequence of FMNL1 and a control sequence were incubated with FreeStyle 293 cell lysate overexpressing srGAP2, photo-crosslinked and washed at high stringency. Immunoblotting with srGAP2 antibody detected phototrapping of srGAP2 by PAX. pBpa in the bait peptides is indicated in blue. (<b>B</b>) Comparative immunoblotting of srGAP2 antigen dot-blot. Different amounts of srGAP2 were spotted onto nitrocellulose membrane and simultaneously immunoblotted with srGAP2 antibody. (<b>C</b>) Identification of srGAP2 by mass spectrometry. The five spots with the highest amount of crosslinked srGAP2 from a duplicate blot of (A) were subjected to MS/MS. 3 unique peptides (underlined) covering 8% of the entire sequence of srGAP2 protein were identified. (<b>D</b>) PAX captures a known interacting protein from mouse brain lysate. Peptide array comprising mGluR5 sequences with a PPXXF motif was incubated with mouse brain lysate, photo-crosslinked and subjected to high stringency washes. Immunoblotting with Homer1 antibody indicates photo-trapping of Homer1 by PAX. pBpa in the bait peptides is indicated in blue.