High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays-2

<p><b>Copyright information:</b></p><p>Taken from "High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays"</p><p>http://www.biomedcentral.com/1471-2164/9/68</p><p>BMC Genomics 2008;9():68-68.</p><p>Published online 6 Feb 2008</p><p>PMCID:PMC2254387.</p><p></p>st and antagonists. : Dose-dependent induction of AR-LBD and AR-NTD interaction by the synthetic agonist R1881, showing a maximal response from 10 nM onwards. : Dose-dependent inhibition of R1881 (10 nM) induced AR-LBD and AR-NTD interaction by two antagonists, medroxyprogesterone acetate (MPA) and hydroxyflutamide (OH-Flu). Quantitative analysis of this inhibition reflects the stronger antagonistic potency of MPA which can achieve maximum inhibition at a concentration of 1 nM as compared to OH-Flu, of which more than 10 nM is required to obtain the same level of inhibition. Data shown represents the mean fluorescence of 6 features per sample, collected from triplicate spots on two identical slides

High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays-1

<p><b>Copyright information:</b></p><p>Taken from "High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays"</p><p>http://www.biomedcentral.com/1471-2164/9/68</p><p>BMC Genomics 2008;9():68-68.</p><p>Published online 6 Feb 2008</p><p>PMCID:PMC2254387.</p><p></p>roteins listed in Additional file and sets of positive (p53+SV40T) and negative (p53+TRAF, single baits and preys) controls were reverse transfected with Hek293T cells in the presence of 10 nM R1881 for 3 days. Each prey-bait combination was printed together with the autofluorescent reporter as single spots per slide with a spot to spot distance of 1.5 mm. Following transfection the fluorescence signals of all 624 features were collected and processed as described in methods. The normalised fluorescence signals obtained from the 480 different bait-prey features (10 baits × 16 preys × 3 replicate slides) are shown separately for the different slides. Sample numbers correspond to the combination of bait and prey as summarized in Additional file . The cutoff value (indicated as red line) resembles the level of reporter expression obtained with the non-interacting control proteins p53+TRAF. This cross-screening using CAPPIA immediately identifies so-called bait-or prey-unspecific false positives as is exemplified by bait B504 (marked with a star). A specific interaction was identified between B487 and P506 corresponding to the AR-LBD and AR-NTD (sample number 23, marked with an arrow)

High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays-3

<p><b>Copyright information:</b></p><p>Taken from "High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays"</p><p>http://www.biomedcentral.com/1471-2164/9/68</p><p>BMC Genomics 2008;9():68-68.</p><p>Published online 6 Feb 2008</p><p>PMCID:PMC2254387.</p><p></p>slides, bait and prey expression plasmids AR-LBD and AR-NTD respectively and the reporter plasmid complexed with transfection reagent were immobilized together in array format. In contrast, on PR slides, each spot contained only the reporter and the AR-NTD prey construct. For Prey-Reporter-stable-bait (PR-SB) experiments a HEK293 cell line with a stable integration of the pBD-LBD plasmid was generated and grown on top of the PR slides. For Prey-Reporter-transient-bait (PR-TB) experiments, suspensions of HEK293T cells were incubated with pBD-LBD plasmid complexed with transfection reagent 5 minutes before adding them to the PR-slides. Finally PRB slides were incubated with non-treated HEK293T cells as described before. All cells were cultured on the slides for 3 days in the presence of 10 nM R1881. : When the data are normalised for differences in transfection efficiencies the results show that all three strategies result in a comparable specific trans-activation of reporter expression following AR-LBD and AR-NTD interaction. Data shown represents the mean fluorescence of 6 features per sample, collected from triplicate spots on two identical slides

High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays-4

<p><b>Copyright information:</b></p><p>Taken from "High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays"</p><p>http://www.biomedcentral.com/1471-2164/9/68</p><p>BMC Genomics 2008;9():68-68.</p><p>Published online 6 Feb 2008</p><p>PMCID:PMC2254387.</p><p></p>ng the Gal4-pZsGreen reporter and plasmids coding for the known interacting p53 (pBD-p53) and SV40-T (pAD-SV40T) hybrid proteins (boxed, line B). As negative control (line A) Gal4-pZsGreen reporter was co-transfected with the non-interacting proteins p53 (pBD-p53) and TRAF (pAD-TRAF). The pBD-NF-κB control plasmid expressing the GAL4 DNA-binding domain fused to the transcription activation domain of NF-κB is used as a positive control to monitor transfection efficiency and reporter performance (line C). A construct expressing EGFP under control of a CMV promoter (line D) is typically printed as a frame at the periphery of the arrays in order to locate the arrayed features. Fig. 1a shows example images of different adherent cell lines transfected using identical microarray slides. : Transfection efficiency as reflected by the level of NF-κB induced reporter expression. Comparable results were obtained for PC-3, HEK293 and HeLa. Data shown are from representative experiments and represent mean fluorescence of 6 features per sample, collected from triplicate spots on two identical slides

Discovery of a Chemical Tool Inhibitor Targeting the Bromodomains of TRIM24 and BRPF

TRIM24 is a transcriptional regulator as well as an E3 ubiquitin ligase. It is overexpressed in diverse tumors, and high expression levels have been linked to poor prognosis in breast cancer patients. TRIM24 contains a PHD/bromodomain offering the opportunity to develop protein interaction inhibitors that target this protein interaction module. Here we identified potent acetyl-lysine mimetic benzimidazolones TRIM24 bromodomain inhibitors. The best compound of this series is a selective BRPF1B/TRIM24 dual inhibitor that bound with a <i>K</i>_D of 137 and 222 nM, respectively, but exerted good selectivity over other bromodomains. Cellular activity of the inhibitor was demonstrated using FRAP assays as well as cell viability data