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Image Search Results
Journal: Molecular pharmacology
Article Title: Site-Directed Mutagenesis of the CC Chemokine Binding Protein 35K-Fc Reveals Residues Essential for Activity and Mutations That Increase the Potency of CC Chemokine Blockade
doi: 10.1124/mol.111.071985
Figure Lengend Snippet: Screening of 35K-Fc mutants by CCR5 DiscoveRx assay. A, dose-response curve showing recruitment of β-arrestin in response to CCR5 activation by RANTES as measured by PathHunter eXpress assay (DiscoveRx). B, RANTES (5 nM) was preincubated for 2 h with the indicated concentration of WT 35K-Fc (●) or MR-Fc (○) and then added to the assay. A and B show mean ± S.E.M. of two technical replicates and are representative of three independent experiments. C, RANTES (5 nM) was preincubated with the indicated 35K-Fc mutant (fixed dose, 15 nM) and then added to PathHunter eXpress CCR5 transfected cells and β-arrestin recruitment measured according to manufacturer’s instructions. Data are shown as the percentage of the response to RANTES alone and are the mean of two independent experiments ± S.E.M., each with two technical replicates for two batches of protein (i.e., four wells per mutant per experiment). Statistical analysis performed by one-way ANOVA and Dunnett’s multiple comparison post test. ***, p < 0.001 relative to WT 35K-Fc.
Article Snippet: Data for all mutants are summarized in . table ft1 table-wrap mode="anchored"
Techniques: Activation Assay, Concentration Assay, Mutagenesis, Transfection
Journal: Molecular pharmacology
Article Title: Site-Directed Mutagenesis of the CC Chemokine Binding Protein 35K-Fc Reveals Residues Essential for Activity and Mutations That Increase the Potency of CC Chemokine Blockade
doi: 10.1124/mol.111.071985
Figure Lengend Snippet: Summary of data for 35K-Fc mutants Values are presented as mean ± S.D. Column 2 summarizes data shown in Fig. 3C and indicates the response to 5 nM RANTES (set as 100%) in a CCR5 DiscoveRx assay after preincubation of 5 nM RANTES with 15 nM 35K-Fc. Column 3 gives the IC 50 (mean of two technical replicates) for the indicated 35K-Fc mutant in a single xCELLigence ECIS screening assay with CCR2-transfected CHO cells responding to 10 nM MCP-1. Columns 4 to 7 show the IC 50 from the number of independent experiments as indicated in parentheses. Statistical analysis was performed by unpaired t test. Columns 4 and 5 summarize the data shown in Fig. 4 . Column 6 gives the IC 50 for the indicated 35K-Fc mutant in a DiscoveRx assay with CCR7-transfected CHO cells responding to 10 nM MIP-3 β (CCL19). Column 7 summarizes the data shown in . The final column indicates the overall rank of the mutants (1, most potent chemokine blockade; 11, least potent).
Article Snippet: Data for all mutants are summarized in . table ft1 table-wrap mode="anchored"
Techniques: Mutagenesis, Screening Assay
Journal: Molecular pharmacology
Article Title: Site-Directed Mutagenesis of the CC Chemokine Binding Protein 35K-Fc Reveals Residues Essential for Activity and Mutations That Increase the Potency of CC Chemokine Blockade
doi: 10.1124/mol.111.071985
Figure Lengend Snippet: Comparison of 35K-Fc WT, R89A, and E143K proteins by DiscoveRx β arrestin assay. A, dose-response curve of beta arrestin recruitment in response to CCR2 activation by MCP-1. B, MCP-1 (3 nM) was preincubated with the indicated concentration of WT 35K-Fc (●), R89A 35K-Fc (■) or E143K 35K-Fc (○) for 2 h then applied to the DiscoveRx assay. C and D, as for A and B, using CCR5 DiscoveRx cells and RANTES as the chemokine ligand. E and F, as for A and B, using CXCR2 DiscoveRx cells and IL-8 as the chemokine ligand. A and B show the mean ± S.E.M. of two technical replicates and are representative of two to three independent experiments. C and D show the mean ± S.E.M. of two technical replicates and are representative of three to four independent experiments. E and F show the mean ± S.E.M. of two technical replicates and are representative of two independent experiments.
Article Snippet: Data for all mutants are summarized in . table ft1 table-wrap mode="anchored"
Techniques: Beta-Arrestin Assay, Activation Assay, Concentration Assay