Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-2

to the human immunoglobulins sequences (imgt). Sequences highlighted in red vary from the amino-acid sequence of scFv C1. Underlined sequences correspond to CDR. Asterisks indicate the missing amino-acid that would generate the usual CDR.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-7

sequence of bacterial pectate lyase that mediates secretion into the periplasmic space; : variable fragment of the heavy chain; : light chain; : 6 histidine-tag; : myc-tag; amber stop codon; : portions of the N- and C-term of phage capside protein pIII; and : primers used for sequencing the Vand Vdomain. Schematic construction of vectors encoding soluble scFv C1 and scFv C1-N1N2. The amber stop codon between the scFv and gene III in pHEN 2 was removed by mutagenesis (middle construct). The C-terminal portion of pIII was removed in the final pHEN C1-N1N2 vector (bottom construct), first by PCR amplification of pHEN C1-pIII, introducing an EcoRI site after N2, and subsequently by cloning the NcoI and EcoRI digested PCR product into the linearized pHEN C1-pIII plasmid at the NotI and EcoRI sites. : 6 histidine-tag; M: myc-tag.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-4

ScFvs were resolved on 12,5% SDS-PAGE, immunoblotted with c-myc antibody, and visualized by enhanced chemiluminescence as described in Methods. (2) scFvs from crude extract were analysed for binding to GST-RhoA and GST-RhoAQ63L protein immobilized on an ELISA plate. Bound scFvs were detected with horseradish peroxydase-labeled anti-c-myc using TMB as substrate. Results are expressed as absorbance at 480 nm. Graphs are representative of 6 experiments, each performed in duplicate.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-0

sequence of bacterial pectate lyase that mediates secretion into the periplasmic space; : variable fragment of the heavy chain; : light chain; : 6 histidine-tag; : myc-tag; amber stop codon; : portions of the N- and C-term of phage capside protein pIII; and : primers used for sequencing the Vand Vdomain. Schematic construction of vectors encoding soluble scFv C1 and scFv C1-N1N2. The amber stop codon between the scFv and gene III in pHEN 2 was removed by mutagenesis (middle construct). The C-terminal portion of pIII was removed in the final pHEN C1-N1N2 vector (bottom construct), first by PCR amplification of pHEN C1-pIII, introducing an EcoRI site after N2, and subsequently by cloning the NcoI and EcoRI digested PCR product into the linearized pHEN C1-pIII plasmid at the NotI and EcoRI sites. : 6 histidine-tag; M: myc-tag.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-1

RhoB (white columns) and GST-RhoBQ63L (black columns) protein immobilized on an ELISA plate. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Helper phage was used as a control. Results are expressed as absorbance at 480 nm. : ratio of absorbance of binding to RhoBQ63L to absorbance of binding to RhoB. : Selectivity of C1 phages on WT and activated Q63L form of Rho. 10clones of C1 (black columns) and control (white columns) phage were analyzed for binding to GST-RhoA, RhoB and RhoC, both wild type (WT) and Q63L forms, immobilized on a glutathione ELISA plate. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Results are expressed as absorbance at 480 nm. Concentrations of GST-Rho proteins in each well were monitored by anti-GST (not shown). The graph is representative for 3 experiments, and each binding assay was performed in duplicate.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-3

100 μM of GDP and GTPγS at 37°C. 10clones of C1 phages were incubated with loaded (GTP or GDP) GST-RhoA-bound beads. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Results are expressed as absorbance at 480 nm. The graph is representative of 2 independents experiments. GST-RhoA, B and C () and GST-RhoB, Rac1 and Cdc42 () were loaded with 200 μM of GDP or GTPγS for 30 min and purified on glutathione ELISA plates. 10clones of C1 phages were incubated in each well. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Results are expressed as absorbance at 480 nm. Amount of GST protein was quantified with goat anti-GST antibody followed by horseradish peroxydase-labeled anti-goat (not shown). The graph is representative of 3 experiments, each binding assay performed in duplicate. : Specific radioactivity binding of [S] GTPγS on RhoB, Rac1 and Cdc42. GST-RhoB, Rac1 and Cdc42 were loaded with 20 nM [S] GTPγS in the presence (non specific binding) or not (total binding) of 200 μM unlabeled GTP for 30 minutes at 37°C and purified on gluthatione ELISA plates. Radioactivity was measured in each well. The difference between the total binding and the non specific binding represent the specific binding. The graph is representative of 2 experiments, each performed in triplicate.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-6

recombinant RhoA and RhoB loaded with either GTP or GDP were incubated with C1-N1N2 fixed on Ni-beads. An irrevelant scFv was used as control. Complexes on beads were resolved by SDS-PAGE and immunobloted with anti-RhoA and anti-RhoB. Western Blot is representative of 2 independent experiments. Immunofluorescence shows that scFv C1-N1N2 specifically binds to activated HeLa cells. Suspension containing scFv C1-N1N2 was incubated with GDP-loaded RhoA(and ) or GTPγS-preloaded RhoA beads (). Twenty-four hours after seeding, HeLa cells were serum-starved for 48 h and activated with 10% SVF and EGF (100 ng/ml) for 1 hour. Cells were fixed, permabilized and incubated with supernatants from scFv C1-N1N2-Rho incubation and anti-c-myc FITC conjugate secondary antibody. ) Non-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GDP-loaded RhoA beads, ) EGF-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GDP-loaded RhoA beads ) EGF-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GTPγS-loaded RhoA beads. () Non-activated HeLa cells incubated with the commercial Rhoa antibody, () EGF-activated HeLa cells incubated with the commercial RhoA antibody, () EGF-activated HeLa cells incubated with irrelevant scFv (anti-tyroglobulin). Pictures are representative of 2 independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Repeated dosing of P2Y13R agonist decreases HDL-C.

<p>Plasma cholesterol (panel A), plasma apoA-I (panel B) and plasma lipoproteins (panel C) concentrations were determined after 2 weeks oral dose of P2Y13R agonist at 100 µg/kg (grey bars) and compared to vehicle treated animals (empty bars). Values for plasma apoA-I vary from 1.5 to 1.85 g/L. *p<0.05. Panel D, Concentration of liver apoA-I was determined by Western-blot quantification (n = 4 mouse/group) using imageJ software. 40 µg of total liver extract (vehicle or CT1007900 at 100 µg/kg) were separated on the same 12.5% SDS-PAGE and probed with goat anti-apoA-I antibody. *p<0.05. Panel E, the ratios of apoA-I/plasma cholesterol concentrations were determined and compared to their respective pre-dose values. Panel F, HDL from C57Bl/6J mouse plasma were separated according to the size of the different HDL particles using the Lipoprint system. The data were expressed as the percentage of difference for each HDL subpopulation set to the HDL population in the pre-dose animals. **p<0.01. Panel G, Determination of cholesterol efflux capacity of mouse plasma (1% v/v) using pre-loaded [³H]-cholesterol-oxLDL macrophages. The results are expressed as a percentage of cholesterol efflux corrected from pre-dose. Values for cholesterol efflux before correction from pre-dose vary from 12–15%. *p<0.05.</p

Increase of HDL recycling following activation of P2Y13R pathway in mice.

<p>C57Bl/6J mice (n = 10) were fasted for 2 h followed by single oral dose of P2Y13R agonist CT1007900 at 3, 30 or 300 µg/kg. Six hours later bile acid content (panel A) and bile cholesterol content (panel B) were evaluated using enzymatic kits. Grey bars represent the amount of bile acid or bile cholesterol per mouse; black bars represent the concentrations of bile acid and bile cholesterol into gallbladder. Panel C, the kinetic of bile acid mobilization in gallbladder induced by CT1007900 at 300 µg/kg (???) by oral gavage (single dosing) using C57Bl/6J mice (n = 5) was evaluated and compared to vehicle treated animals (○). *p<0.05, **p<0.01, ***p<0.0005. Bile acid content of liver (panel D) was evaluated using enzymatic kit. * p<0.05, **p<0.01. Plasma cholesterol (panel E) and plasma apoA-I (panel F) concentrations were determined at different time points after single oral dose of P2Y13R agonist at 100 µg/kg () and compared to vehicle treated animals (○). Values in pre-dose groups for plasma cholesterol vary from 0.85 to 0.95 g/L, and 1.1 to 1.3 g/L for plasma apoA-I. Panel G, C57Bl/6J mice (n = 5) were intravenously injected with [³H]-cholesterol-labelled mouse HDL (10 µCi/mouse) and CT1007900 (10 nmole/kg or 4 µg/kg). Radioactivity present in the liver was determined 2 hours later. **p<0.01. Panel H, C57Bl/6J mice (n = 5) were dosed (single dosing) with CT1007900 (100 µg/kg) and intravenously injected with [³H]-cholesterol-labelled mouse HDL (10 µCi/mouse). Feces from individual mouse were collected for 6 h and extracted for cholesterol (empty bars) and bile acid content (grey bars) and the radioactivity was determined by scintillation counting. *p<0.05.</p

Effect of CT1007900 on atherosclerotic plaque progression in carotids of apoE^−/− mice.

<p>Pannel A. ApoE^−/− mice (n = 7) were ligatured on the upper part of the left carotids. On the day of the surgery the animals were placed on a HCD and given an oral gavage of vehicle or increasing doses of compound CT1007900. Ligatured carotids were lipid extracted in 2∶1 chloroform/methanol and the concentrations of total cholesterol were measured by HPLC. **p<0.01. Panel B, C and D: longitudinal sections of apoE^−/− mice ligated carotids were analyzed by hematoxylin eosin staining, Oil Red O staining and CD-68 antibody staining respectively. *p<0.05. Panel E: Liver unesterified cholesterol determination. **p<0.01. Panel F. ApoE^−/− mice (n = 10) were infected with 5×10⁹ adenoviral particles coding empty vector (mock) or vector encoding P2Y13R shRNA, 3 days before the ligation of the left carotid. On the day of surgery the animals were placed on a Western diet and also given oral gavage of vehicle or compound CT1007900 at 100 µg/kg, once a day for 2 weeks. Ligatured carotids were lipid extracted in 2∶1 chloroform/methanol. The concentrations in total cholesterol were measured by HPLC. **p<0.01.</p