Post-selection and further confirmations. (A) Interaction analyses by pull-down assay. Pull-down assays of all 12 genes listed in Table 1 using C-terminally labeled proteins (Miyamoto-Sato et al. 2000; Doi et al. 2002) were performed with (+) or without (–) Fos as bait. The results of 1% SDS-PAGE for seven genes are shown. (Lanes 1–3) Translation product (input), supernatant, eluate (output), respectively. (B) Enrichment analyses with real-time PCR. All 12 genes listed in Table 1 were analyzed in each round's pool (1st to 4th) of Experiment 2, and results for six genes, Jun (○), Jund1 (•), Rit2 (□), Optn (▪), Snapc5 (▵), C130020M04Rik (▴), are shown. (Ini) Initial library. β-Actin (×) is a negative control. (C) Further confirmation of Troarfips. Coimmunoprecipitation (IP) of Fos and Troarfip1 in COS-1 cells was performed as described in “Methods”. (Lanes 1–3 [lysate]) Negative control (background), coexpression of Fos and Jun, and coexpression of Fos and Flag-Troarfip1, respectively. (Lanes 4–6 [IP]) IP with Jun using coexpression of Fos and Jun, IP with Troarfips using coexpression of Fos and Flag-Troarfip1, and IP with Flag using coexpression of Fos and Flag-Troarfip1, respectively. (D) Further confirmation of Snapc5. Pull-down assays of Snapc5 in Table 1 using C-terminal labeled proteins were performed with (+) or without (–) Jun as bait, followed by analysis on 15% SDS-PAGE. (Lanes 1–3) Translation product (input), supernatant, eluate (output), respectively.
