To confirm whether protein-protein interactions were correct [31]. The SOS recruitment system, which is based on the rescue of rasmediated signal transduction in a cdc25-2 temperature-sensitive yeast strain, facilitates interactions between partner proteins in the cytoplasm as opposed to in the nucleus with the conventional yeast two-hybrid system [32]. Another approach to increase the specificity of protein interactions is based on the concept of a multiple reporter gene system utilizing a triple reporter assay with URA3, MEL1 and LacZ genes [33]. A more recently developed approach to reduce false-positive reactions is based on the concept of permutated fusion proteins, which contain a mixture of N- and C-terminal bait fusion proteins [34]. Finally, because the cells used in the detection systems can activate the transcription of reporter genes by their internal proteins, the method of interrogating protein-protein interactions within the Golgi center, where internal transcriptional activation is reduced to a negligible background level, has been utilized [35]. All these KS 176 approaches were designed to improve the specificity of transcriptional activation as well as the specificity of bait-and-prey interactions. They did not, however, address the problems related to the cDNA library construction with 59-UTR-based frame shifts that may affect a large proportion of the expressed proteins. The in-frame cDNA library we have described here enables the effective removal of 59-UTRs from the constructs, thereby facilitating the expression of correct proteins. The PCR approach utilizing primers with Kozak sequences permitted the capture of all the genes in the library, increasing the chance of identifying correct protein interactions. This system is anticipated to have wide applications in the detection of protein interactions utilizing genome-wide expression libraries across mammalian and nonmammalian species.Materials and Methods Cell Line and Human TissueAll human tissues were collected with written informed consent under protocols approved by The University of Texas MD Anderson Cancer Center Institutional Review Board for this study, and the samples were analyzed anonymously. The HEK293T cell line was obtained from the American Type Culture Collection and was maintained in Dulbecco’s modified Eagle’sIn-Frame cDNA LibraryFigure 2. Predicted expression of CRABP2 and PGAM1 proteins by the SPDP biological activity constructs with and without 59-UTRs. (A) Comparison of CRABP2 expression constructs with and without the CRABP2 59-UTR. (B) Comparison of PGAM1 expression constructs with and without the PGAM1 59-UTR. doi:10.1371/journal.pone.0052290.gmedium supplemented with 10 fetal bovine serum in a humidified incubator under an atmosphere of 5 CO2 in air. Under an approved institutional protocol, normal human urothelial cells were obtained from ureters attached to nephrectomy specimens performed for excision of renal cell carcinomaswithout any evidence of the tumor involvement of the renal calyxes, pelvis, or ureter. The urothelial cell suspensions were prepared by scraping the urothelial surface and resuspending the cells in phosphate-buffered saline (PBS) as previously described [7].In-Frame cDNA LibraryFigure 3. Identification of the CRABP2 and PGAM1 proteins as ARL11-binding partners using the in-frame cDNA expression library. (A) Western blot analysis of N-terminal YFP1-tagged 12926553 fusion proteins expressed by the constructs with and without 59-UTRs in HEK-293T cells. Expr.To confirm whether protein-protein interactions were correct [31]. The SOS recruitment system, which is based on the rescue of rasmediated signal transduction in a cdc25-2 temperature-sensitive yeast strain, facilitates interactions between partner proteins in the cytoplasm as opposed to in the nucleus with the conventional yeast two-hybrid system [32]. Another approach to increase the specificity of protein interactions is based on the concept of a multiple reporter gene system utilizing a triple reporter assay with URA3, MEL1 and LacZ genes [33]. A more recently developed approach to reduce false-positive reactions is based on the concept of permutated fusion proteins, which contain a mixture of N- and C-terminal bait fusion proteins [34]. Finally, because the cells used in the detection systems can activate the transcription of reporter genes by their internal proteins, the method of interrogating protein-protein interactions within the Golgi center, where internal transcriptional activation is reduced to a negligible background level, has been utilized [35]. All these approaches were designed to improve the specificity of transcriptional activation as well as the specificity of bait-and-prey interactions. They did not, however, address the problems related to the cDNA library construction with 59-UTR-based frame shifts that may affect a large proportion of the expressed proteins. The in-frame cDNA library we have described here enables the effective removal of 59-UTRs from the constructs, thereby facilitating the expression of correct proteins. The PCR approach utilizing primers with Kozak sequences permitted the capture of all the genes in the library, increasing the chance of identifying correct protein interactions. This system is anticipated to have wide applications in the detection of protein interactions utilizing genome-wide expression libraries across mammalian and nonmammalian species.Materials and Methods Cell Line and Human TissueAll human tissues were collected with written informed consent under protocols approved by The University of Texas MD Anderson Cancer Center Institutional Review Board for this study, and the samples were analyzed anonymously. The HEK293T cell line was obtained from the American Type Culture Collection and was maintained in Dulbecco’s modified Eagle’sIn-Frame cDNA LibraryFigure 2. Predicted expression of CRABP2 and PGAM1 proteins by the constructs with and without 59-UTRs. (A) Comparison of CRABP2 expression constructs with and without the CRABP2 59-UTR. (B) Comparison of PGAM1 expression constructs with and without the PGAM1 59-UTR. doi:10.1371/journal.pone.0052290.gmedium supplemented with 10 fetal bovine serum in a humidified incubator under an atmosphere of 5 CO2 in air. Under an approved institutional protocol, normal human urothelial cells were obtained from ureters attached to nephrectomy specimens performed for excision of renal cell carcinomaswithout any evidence of the tumor involvement of the renal calyxes, pelvis, or ureter. The urothelial cell suspensions were prepared by scraping the urothelial surface and resuspending the cells in phosphate-buffered saline (PBS) as previously described [7].In-Frame cDNA LibraryFigure 3. Identification of the CRABP2 and PGAM1 proteins as ARL11-binding partners using the in-frame cDNA expression library. (A) Western blot analysis of N-terminal YFP1-tagged 12926553 fusion proteins expressed by the constructs with and without 59-UTRs in HEK-293T cells. Expr.