Proxiplate 384 plus plates

Glucose agar plates 10 cm CM minus Trp/Ura from Teknova (C3260), Streakers wooden sticks from Biolog (3026), CM galactose broth Trp^-/Ura^- from Teknova (C9130), CM glucose broth Trp^-/Ura^- from Teknova (C8240), vented 50 mL conical flasks form TRP (87050), 96 well U-Bottom plates from Falcon (353077), 96 well plate 2 mL PP from Thomson Instrument company (931130), Blocker Casein in PBS from Thermo (QD216041), 2xYT from Teknova (2Y1080), AF647 anti-M-13 conjugated in-house according to manufacturer recommendation, Anti-V5 antibody from Invitrogen (46–1157), FITC-anti mouse IgG2a form Biolegend (407106), PE-Goat anti-mouse IgG2b from Southern Biotech (1090-09S), Human IL-12/IL-23 P40 Mab Clone1645 from R&D Systems (Mab 6091–500). Peptide synthesis was outsourced to CPC Scientific. Avi-tagged IL-23 and FLAG-tagged IL-23R were expressed and purified in house following standard Molecular Biology procedures. Alpha-screen Streptavidin Donor beads (#509048876) and AlphaLISA® Acceptor beads conjugated to anti-FLAG antibody (#AL112M) were purchased from Perkin Elmer. ProxiPlate-384 Plus plates (#6008289) for AlphaLISA assays were also obtained from Perkin Elmer. PathHunter® U2OS IL-23R/IL-12RB1 Dimerization Cell Line was purchased from DiscoverX (#93-1007C3). 96-well half area plates used in the dimerization assay were from Costar (#3688).

ALPHAScreen was performed as previously described^{16 (link)–18 (link)}, using the cMyc detection kit and Proxiplate-384 Plus plates (PerkinElmer). The plates were incubated for 45 min at room temperature, followed by the addition of streptavidin-coated donor beads and incubation in the dark for 45 min at room temperature. The ALPHAScreen signal was measured on an Enivision Plate Reader (PerkinElmer) using manufacturer’s recommended settings. Data from three independent experiments was analyzed in GraphPad Prism version 6.0. The luminescence intensities plotted are averages over three independent expressions/experiments where for each experiment, the signal intensity from a negative control, GFP alone in solution was substracted. An average of all the normalized data for both configuration of each protein pair (GFP-protein A/protein B-Cherry or GFP protein B/protein A-Cherry) was calculated to provide the binding index where values above 2000 were considered background. From previous experiments^{16 (link)–18 (link)}, a threshold of above 2000 (background) selected positive interactions and was therefore employed in these studies.