Table 1. Thirty-five selected proteins Gene symbol Nrbf2 4732436F15Rik c-Maf* SNAP19 Fos* Mapre3 Cspg6 Kif5A (region C) 9130229H14Rik Jun* Mapk8ip3 Creb3 Kif5B (region C) Kif5C (region C) Nef3 Eef1d Atf7* Atf4* Cutl1 Jdp2* Ofd1 GFAP Kif5C (region N) Psmc5 Atf3* Kif5B (region N) B130050I23Rik Cebpg 1200008A14Rik Myh11 Tax1bp1 Myt1 Fosl2* Tef Cebpz + + - + + + + + + + + - - + + + - + - + - + + - + - + - + - - - - - - NM_025307.2 XM_143418.3 S74567.1 NM_025925.1 NM_010234.2 NM_133350.1 NM_007790.2 NM_008447.2 XM_135706.4 NM_010591.1 NM_013931.1 XM_131375.2 NM_008448.2 NM_008691.1 NM_008449.2 NM_029663.1 NM_146065.1 NM_009716.1 NM_009986.2 NM_030887.2 NM_177429.2 NM_010277.1 NM_008449.2 NM_008950.1 NM_008448.2 NM_007498.2 NM_153536.2 XM_133383.2 NM_028915.1 NM_013607.1 NM_025816.1 NM_008665.2 NM_008037.3 NM_017376.2 NM_009882.1 8.255 8.134 7.796 7.734 7.720 7.714 7.577 7.491 7.487 7.411 7.282 7.172 7.141 7.129 7.106 7.083 6.999 6.991 6.850 6.768 6.692 6.551 6.098 5.961 5.937 5.574 5.213 5.122 4.623 4.343 4.176 4.084 3.935 3.467 3.012 Cloning & Sequencing Accession no. Signal ratio Locus on mRNA sequence (base) Abundance ratio in the initial library (%) Abundance ratio in the 5th round library (%) Enrichment 651…950 2051…2300 1805…2004 1…250 501…800 601…950 2151…2700 2251…2900 51…400 1701…1950 1351…1800 601…800 2801…3200 951…1300 2701…3200 1301…1750 1051…1300 1001…1300 301…500 451…700 1752…2001 901…1100 1201…1450 51…300 1301…1550 401…950 1151…1450 401…750 1501…1750 3251…3650 501…750 3251…3550 401…750 851…1000 1451…1750 5.7E-4 1.1E-4 6.4E-5 1.6E-3 7.4E-5 9.2E-4 4.1E-4 3.3E-3 1.6E-3 1.4E-4 7.3E-4 3.3E-4 1.3E-3 8.2E-3 2.2E-3 5.5E-3 2.7E-5 5.4E-4 1.4E-3 7.1E-4 1.4E-4 1.1E-3 1.2E-2 3.5E-3 6.4E-4 1.1E-7 1.9E-4 3.5E-5 3.6E-4 1.4E-4 2.7E-3 1.1E-5 2.1E-5 1.3E-4 1.2E-4 Tiling Arrays for mRNA Display 4.8E-1 1.5E+0 5.2E-1 2.2E+1 1.5E+0 1.4E+0 1.0E+0 4.3E+0 1.2E+0 4.3E-1 5.3E-1 2.6E-3 1.2E-1 1.5E+0 6.4E+0 2.3E+0 1.4E-1 1.9E+0 3.2E-1 2.1E+0 2.6E-1 8.8E-2 5.2E+0 6.4E-2 1.5E-1 2.0E-2 1.5E-2 2.7E-3 2.1E-1 2.9E-4 1.0E-2 1.3E-4 1.4E-2 1.1E-3 3.7E-4 840 14,000 8,000 14,000 20,000 1,600 2,500 1,300 730 3,100 720 8 95 190 2,900 420 5,000 3,500 230 3,000 1,800 77 450 19 230 80 78 600 2 4 8 3 12 670 180,000 *Previously reported interactors with Jun. doi:10.1371/journal.pone.0001646.t001 Verification of the newly found protein-protein interactions To confirm the physical association of the 11 newly discovered candidates with Jun, we first performed in vitro pull-down assay. Seven of the 11 tested candidates exhibited specific interaction with the bZIP domain of Jun (Figure S2 online). The affinity of the remaining four proteins, i.e., Cutl1, Myh11, Tax1bp1 and Cebpz, for Jun may be weaker, because their enrichment ratios (excluding that of Cutl1) in the IVV screening were lower than those of others (Table 1). Thus, we next employed the surface plasmon resonance (SPR) method, a highly sensitive analysis tool for protein interactions. In this case, all of the candidates except Tax1bp1 interacted with Jun in a concentration-dependent fashion (Figure S3 online). Although most of the above tested interactions seem to be very weak, we considered that the interactions are true positives, because all of the candidates except Cebpz contain leucine-heptad repeats in the selected regions, and such repeats are an important motif for heterodimerization with Jun. Further experiments in vivo will be required to examine the physiological roles of these interactions. Discussion To evaluate the quality of the interaction data, coverage and accuracy were calculated as follows. Jun interacts with many bZIP superfamily proteins and structurally unrelated transcription PLoS ONE | www.plosone.org 4 February 2008 | Volume 3 | Issue 2 | e1646