Contexts in source publication

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... detect protein-protein binding 33 . Unfortunately, our initial system did not yield a phosphorylationdependent transcriptional response, so we complemented it with inducible plasmids-each harboring a different system component-to identify proteins with suboptimal expression levels ( Figure 1B). Interestingly, secondary induction of Src increased luminescence, an indication that insucient substrate phosphorylation and/or weak substrate-SH2 binding depressed GOI expression in our base system. ...

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... modied this system by swapping in different substrate domains, by adding mutations to the SH2 domain that enhance its anity for phosphopeptides 34 , and by removing the gene for Src, a modication that allowed us to control its expression exclusively from a second plasmid. With this conguration, induction of Src increased luminescence most prominently for the MidT substrate ( Figure 1C), and simultaneous induction of both Src and PTP1B prevented that increase-an indication of intracellular PTP1B activity ( Figure 1D). We nalized the MidT system by incorporating genes for PTP1B and Src, by adjusting promoters and ribosome binding sites to amplify its transcriptional response further ( Figure 1D, S1-2), and by adding a gene for spectinomycin resistance (SpecR) as the GOI. ...

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... modied this system by swapping in different substrate domains, by adding mutations to the SH2 domain that enhance its anity for phosphopeptides 34 , and by removing the gene for Src, a modication that allowed us to control its expression exclusively from a second plasmid. With this conguration, induction of Src increased luminescence most prominently for the MidT substrate ( Figure 1C), and simultaneous induction of both Src and PTP1B prevented that increase-an indication of intracellular PTP1B activity ( Figure 1D). We nalized the MidT system by incorporating genes for PTP1B and Src, by adjusting promoters and ribosome binding sites to amplify its transcriptional response further ( Figure 1D, S1-2), and by adding a gene for spectinomycin resistance (SpecR) as the GOI. ...

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... this conguration, induction of Src increased luminescence most prominently for the MidT substrate ( Figure 1C), and simultaneous induction of both Src and PTP1B prevented that increase-an indication of intracellular PTP1B activity ( Figure 1D). We nalized the MidT system by incorporating genes for PTP1B and Src, by adjusting promoters and ribosome binding sites to amplify its transcriptional response further ( Figure 1D, S1-2), and by adding a gene for spectinomycin resistance (SpecR) as the GOI. The nal plasmid-borne detection system required the inactivation of PTP1B to permit growth at high concentrations of antibiotic ( Figure 1E). ...

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... nalized the MidT system by incorporating genes for PTP1B and Src, by adjusting promoters and ribosome binding sites to amplify its transcriptional response further ( Figure 1D, S1-2), and by adding a gene for spectinomycin resistance (SpecR) as the GOI. The nal plasmid-borne detection system required the inactivation of PTP1B to permit growth at high concentrations of antibiotic ( Figure 1E). ...

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... It appears to adopt multiple bound conformations (i.e., the electron density indicates regions of disorder; Figure S8). This behavior, which is supported by molecular dynamics simulations ( Figure S10), is not unusual for hydrocarbon moieties, which can exhibit enhanced mobility in protein binding pockets 52 . ...

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... structural analogue of amorphadiene has a carboxyl group in a position likely to interfere with binding to the hydrophobic cleft ( Figure 3C). The IC 50 of this molecule was eight-fold higher than that of amorphadiene, a reduction in potency consistent with its crystallographic pose ( Figure 3D , Figure S11L). Second, we examined competition between amorphadiene and two inhibitors that bind to the active site: (i) TCS401, which causes the WPD loop to adopt a closed conformation, and (ii) orthovanadate, which does not. ...

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... we examined the sensitivity of amorphadiene andbisabolene to the removal of the 7 helix, which is proximal to the C-terminal allosteric site. This truncation reduced the potency of both terpenoids by four-to ve-fold; the 7 helix thus appears to contribute to terpenoid binding ( Figure S11). Fourth, we assessed the selectivity of amorphadiene andbisabolene for PTP1B over TC-PTP, its closest homolog (the two catalytic domains share 68% sequence identity 53 ). ...

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... we assessed the selectivity of amorphadiene andbisabolene for PTP1B over TC-PTP, its closest homolog (the two catalytic domains share 68% sequence identity 53 ). Both molecules inhibited TC-PTP ve-to six-fold less potently than PTP1B ( Figure 3G, Figure S11A-K), a nding consistent with binding to the poorly conserved allosteric site. This selectivity may seem modest, but it matches or exceeds the selectivities of most pre-optimized inhibitors (e.g., including benzbromarones) and is rare for unfunctionalized hydrocarbons 54 . ...

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... is a receptor tyrosine kinase that undergoes PTP1B-mediated dephosphorylation from the cytosolic side of the plasma membrane (PTP1B, in turn, localizes to the endoplasmic reticulum of the cell). Both molecules increased IR phosphorylation over a negative control ( Figure 3G, Figure S13). We checked for off-target contributions to this signal, in turn, by repeating the ELISA with equivalent concentrations of dihydroartemisinic acid and -bisabolol, which exhibit potencies that are eight-and twenty-fold lower than those of amorphadiene and -bisabolene, respectively ( Figure S11). ...

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... molecules increased IR phosphorylation over a negative control ( Figure 3G, Figure S13). We checked for off-target contributions to this signal, in turn, by repeating the ELISA with equivalent concentrations of dihydroartemisinic acid and -bisabolol, which exhibit potencies that are eight-and twenty-fold lower than those of amorphadiene and -bisabolene, respectively ( Figure S11). To our satisfaction, both molecules led to a reduction in signal consistent with their reduced potencies for PTP1B. ...

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... PTPs can promote IR dephosphorylation; SHP1 and SHP2 are important examples [55][56][57] . To assess the potential contribution of these enzymes to the increase in IR phosphorylation observed in our ELISA experiments, we measured their inhibition by amorphadiene and -bisabolene ( Figure S12). Amorphadiene inhibited SHP2 three-fold less potently than PTP1B; its inhibition of SHP1 was too weak to measure. ...

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... used a custom MATLAB script to process all raw kinetic data. This script removed all concentration values that fell outside of either (i) the range of our standard curve (absorbance/uorescence vs. μM; Figure S21) or (ii) the initial rate regime (>10% of the pNPP or 4-MUP concentration used in the assay). When this step reduced kinetic dataset to fewer than ten points, we re-measured those datasets to collect at least ten. ...

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... measured the inuence of inhibitors on insulin receptor (IR) phosphorylation by using an IR-specic ELISA ( Figure S13A). Briey, we starved cells for 48 hours in FBS-free media and incubated the with inhibitors (all at 3% DMSO) for 10 minutes. ...

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... measured IR phosphorylation in subsequent dilutions of the 60 mg/ml samples with the PathScan® Phospho-Insulin Receptor β (panTyr) Sandwich ELISA Kit (Cell Signaling Technology; #7082). To identify biologically active concentrations of -bisabolene and amorphadiene, we screened several concentrations and chose those that gave the highest signal (405 μM for -bisabolene and 930 μM for amorphadiene); similar concentrations of weak inhibitors did not yield a detectable signal ( Figure S13B,C). ...