Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: In vitro susceptibilities of planktonic S. mutans UA159

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: In Vitro

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: In Vitro

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: Comparative killing kinetics of CLP-4. S. mutans UA159 cultures at a cell density of 6 × 105 CFU/ml were challenged with 5, 10, and 25 μg/ml CLP-4 under conditions of active growth in CDM supplemented with 0.5% (wt/vol) glucose (A) and against growth-arrested cells in CDM lacking any carbon source (B). Samples at time zero were enumerated prior to peptide treatment. Data shown are the means and standard deviations of three biological replicates from three independent experiments.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: CLP-4 prevents S. mutans biofilm formation. (A) Biofilms inoculated with 2 × 107 CFU/ml were grown for 24 h in the presence of CLP-4, chlorhexidine, or erythromycin at concentrations ranging between 0.6× and 2× their respective MICs. Biofilm formation was quantified using crystal violet staining and expressed in percentage relative to untreated control. Shown are the means and standard deviations of three biological replicates from three independent experiments. *, P < 0.05; ***, P < 0.001 compared to untreated control. (B) Corresponding growth curve kinetics showing the MIC of CLP-4 on S. mutans UA159.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: Staining, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: Effects of CLP-4 on preformed biofilms. S. mutans UA159 biofilms were established for 24 h and then treated with increasing concentrations (1× to 10× the MIC) of CLP-4, chlorhexidine, or erythromycin. (A) Antibiofilm activities were assessed by quantifying the cell viability of treated biofilms by colony enumeration on agar plates. The means and standard deviations of three biological replicates from three independent experiments are shown. **, P < 0.01; ***, P < 0.001 compared to untreated control. (B) Biofilms treated with 10× the MICs for each antimicrobial were fluorescently labeled using the LIVE/DEAD BacLight viability stain and visualized by confocal laser scanning microscopy. Shown are the top-down three-dimensional (3D) volume rendering of biofilms at a total magnification of ×400. Bottom images represent optical planes in the xz, and vertical thin images represent yz dimensions. Membrane-compromised bacteria are stained red with propidium iodide, while intact bacteria are stained green with SYTO 9. Areas highlighted by dashed lines indicate regions of interest (ROIs) viewed at a higher magnification. Dimensions shown are 387.5 μm by 387.5 μm by 16 μm. (C) ROIs are presented at ×2,300 magnification. Dimensions shown are 68.1 μm by 68.1 μm by 16 μm.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: Control, Labeling, Staining, Confocal Laser Scanning Microscopy, Membrane, Bacteria

Journal: Acta Crystallographica. Section F, Structural Biology Communications

Article Title: Crystal structure of the aromatic-amino-acid aminotransferase from Streptococcus mutans

doi: 10.1107/S2053230X18018472

Figure Lengend Snippet: Macromolecule-production information

Article Snippet: Macromolecule-production information is summarized in Table 1 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Source organism S. mutans DNA source S. mutans strain UA159 (ATCC 700610) Forward primer 5′-CGC GGATCC ATGGATTTGAGTAAACGTTTTA-3′ Reverse primer 5′-CCG CTCGAG TTAGTCTGCATATTGCTCC-3′ Cloning vector pGEX-6P-1 Expression vector pGEX-6P-1 Expression host E. coli strain BL21 (DE3) Complete amino-acid sequence of the construct produced GPMDLSKRFNKNLNKIEVSMIRQFDQSISDIPDVLKLTLGEPDFATPKHIKEAAKRAIDADESHYTGMAGLLALRQAASAFVKEKYHLTYNPDNEILVTIGATEALSASLTAILEPGDKVLLPAPAYPGYEPVVNLVGAEVVEIDTRSNDFVLTPEMLEEAILKEGEALKAVILNYPTNPTGVTYSRQQIKNLAEVLKKYPIFVISDEVYAELTYTGESHVSIAEYLPDQTILISGLSKSHAMTGWRLGLIFAPAVLTAQLIKSHQYLVTAATTSVQFAAIEALTNGKDDALPMKEEYIKRRDYIIEKMEAMKFKIIKPDGAFYIFAKIPVAQGQDSFKFLQDFAKEKAVAFIPGVAFGKYGEGYLRISYAASMETIKEAMKRLKEFMEQYAD Open in a separate window caption a8 Macromolecule-production information 2.2.

Techniques: Cloning, Plasmid Preparation, Expressing, Sequencing, Construct, Produced

Journal: Ontario Health Technology Assessment Series

Article Title: Cell-Free Circulating Tumour DNA Blood Testing to Detect EGFR T790M Mutation in People With Advanced Non–Small Cell Lung Cancer: A Health Technology Assessment

doi:

Figure Lengend Snippet: EGFR Mutation Kits Approved by Health Canada

Article Snippet: Two kits are approved by Health Canada and are used to detect the EGFR mutation in blood samples ( ). table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Description Characteristic Therascreen EGFR Plasma RGQ PCR Kit Cobas EGFR Mutation Test Version 2 Manufacturer Qiagen Roche Licence number 97247 98447 Type Test kit Test kit Device class 3 3 First issue date 2016-07-08 2017-01-24 Detection method Analogue Semi-quantitative Detects 21 mutations Analogue Semi-quantitative Detects 42 mutations Open in a separate window Abbreviations: EGFR , epidermal growth factor resistance; PCR, polymerase chain reaction; RGQ, rotor-gene Q. EGFR Mutation Kits Approved by Health Canada Ontario and Canadian Context In Ontario, liquid biopsy is being used to detect the presence of the EGFR T790M resistance mutation in patients with NSCLC who are no longer responding (i.e., their cancer has progressed) to first- (gefitinib and erlotinib) or second-generation (afatinib) EGFR -TKI therapy.

Techniques: Mutagenesis, Clinical Proteomics

Journal: Ontario Health Technology Assessment Series

Article Title: Cell-Free Circulating Tumour DNA Blood Testing to Detect EGFR T790M Mutation in People With Advanced Non–Small Cell Lung Cancer: A Health Technology Assessment

doi:

Figure Lengend Snippet: EGFR T790M Testing Costs

Article Snippet: Two kits are approved by Health Canada and are used to detect the EGFR mutation in blood samples ( ). table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Description Characteristic Therascreen EGFR Plasma RGQ PCR Kit Cobas EGFR Mutation Test Version 2 Manufacturer Qiagen Roche Licence number 97247 98447 Type Test kit Test kit Device class 3 3 First issue date 2016-07-08 2017-01-24 Detection method Analogue Semi-quantitative Detects 21 mutations Analogue Semi-quantitative Detects 42 mutations Open in a separate window Abbreviations: EGFR , epidermal growth factor resistance; PCR, polymerase chain reaction; RGQ, rotor-gene Q. EGFR Mutation Kits Approved by Health Canada Ontario and Canadian Context In Ontario, liquid biopsy is being used to detect the presence of the EGFR T790M resistance mutation in patients with NSCLC who are no longer responding (i.e., their cancer has progressed) to first- (gefitinib and erlotinib) or second-generation (afatinib) EGFR -TKI therapy.

Techniques: Ubiquitin Proteomics, DNA Extraction, Sequencing, Mutagenesis, Digital PCR, Reverse Transcription Polymerase Chain Reaction, Next-Generation Sequencing

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: A P4HA2 interacts with KIF7. HEK293T cells were transfected with Flag-tagged P4HA2. Cell lysates were prepared and subjected to immunoprecipitation (IP) with M2 anti-Flag beads. The immunoprecipitates were analyzed by Western blotting (WB) with anti-Flag and KIF7 antibodies. B KIF7 interacts with P4HA2. HEK293T cells were transfected with Flag-tagged KIF7. Cell lysates were prepared and subjected to Flag IP. The immunoprecipitants were analyzed by WB with anti-Flag and P4HA2 antibodies. C Schematic representation of the SHH signaling pathway. SAG is a chlorobenzothiophene-containing Hh pathway agonist and binds to the SMO heptahelical bundle in a manner that antagonizes cyclopamine action. SAG activates SMO, leading to the release of GLI1 by SUFU, and the Hh signaling pathway is then activated. D – I Knockout of P4HA2 causes suppression of Hedgehog signal transduction. NIH/3T3 ( D – F ) and OP9 ( G – I ) P4HA2 knockout (KO) or vehicle cells were treated with (+) or without (−) 200 nM SAG. The Hh targeted genes, Gli1 ( E , H ) and Ptch1 ( F , I ), were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Data are shown as the mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All experiments were repeated three times independently.

Article Snippet: The following antibodies were used for western blotting (WB), immunofluorescence (IF): PAN-OH (home-made; 1:1,000 for WB); P4HA2 (13759-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC, 1:200 for IF); P4HA1 (12658-1-AP, rabbit, Proteintech; 1:1,000 for WB); KIF7 (24693-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC); SUFU (26759-1-AP, rabbit, Proteintech; 1:2,000 for WB); Flag (clone M2, F1804, mouse, Sigma-Aldrich; 1:10,000 for WB, 1:200 for IF); His (clone OGHis, D291-3, mouse, Medical & Biological Laboratories; 1:5,000 for WB); V5 (AB3792, rabbit, Merck Millipore; 1:2,000 for WB); GLI1 (clone A-7, sc-515781, mouse, Santa Cruz; 1:1,000 for WB, 1:200 for IHC); GLI2 (18989-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC) ; GAPDH (clone 1E6D9, 60004-1-Ig, mouse, Proteintech; 1:10,000 for WB); Arl13b (clone N295B/66, 75-287, mouse, NeuroMab; 1:200 for IF); Cy3 AffiniPure Goat Anti-Mouse IgG (H + L) (115-165-003, Jackson ImmunoResearch; 1:10,000 for IF); α-SMA (#34105, Cell Signaling Technology, 1:100 for IF) Light chain specific Mouse Anti-Rabbit IgG (211-032-171, Jackson ImmunoResearch; 1:10,000 for WB); Light chain specific Goat Anti Mouse IgG (115-035-174, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Rabbit IgG, Fc fragment specific (111-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Donkey anti-mouse IgG (H&L) (715-035-150, Jackson ImmunoResearch; 1:10,000 for WB); Goat Anti-Rabbit IgG (H + L) (111-035-003, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific (115-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Smoothened Agonist (SAG), Selleck, Cat# S7779; Fetal bovine serum, Thermo Fisher, Cat# 16140071; Penicillin/streptomycin, Thermo Fisher, Cat# 15140122; RPMI 1640, Thermo Fisher, Cat# 11875093; DMEM, Thermo Fisher, Cat# 11995065; Puromycin Dihydrochloride, Sigma, Cat# P8833; Trypsin 0.25% EDTA, Thermo Fisher, Cat# 25200072; Polyethylenimine Hydrochloride (MW 40,000), Polysciences, Cat# 24765-1; Polybrene Transfection Reagent, Sigma, Cat# TR-1003-G; SHH protein, Yun Zhao Laboratory; Anti-V5-tag mAb-Magnetic Beads, Medical & Biological Laboratories, Cat# M215-11; NTI-FLAG® M2 Affinity Gel, Sigma, Cat# F2426; Ni-NTA Agarose, QIAGEN, Cat# 30230; HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01/02; ChamQ SYBR qPCR Master Mix, Vazyme, Cat# Q311-02.

Techniques: Transfection, Immunoprecipitation, Western Blot, Knock-Out, Transduction, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: A Schematic of full-length KIF7 proteins. The KIF7 protein encodes a 1343 aa protein with a kinesin motor domain, GLI-binding domain (BD), Nephoronophthisis-1-interacting domain (NPHP1-ID) and a Structural Maintenance of Chromosomes (SMC) domain representing the ATPase activity. Various truncated mutation constructs of KIF7-Flag are shown schematically. B Mapping the interaction domain of KIF7 with P4HA2. HEK293T cells were transfected with constructs shown as ( A ). Cell lysates were prepared and subjected to immunoprecipitation (IP) with M2 anti-Flag beads. C Mapping the minimum interaction domain of KIF7 with P4HA2. HEK293T cells were transfected with constructs “a”, “f” and “g” mutants. Cell lysates were prepared and subjected to Flag IP. D , E Endogenous co-immunoprecipitated P4HA2 and KIF7 interact with each other separately in HEK293T cells. Endogenous KIF7 and P4HA2 were precipitated from a HEK293T cells extract and analyzed by immunoblotting. F P4HA2 interacts with SUFU and GLI2. HEK293T cells were transfected with Flag-tagged P4HA2. Cell lysates were subjected to Flag IP. The immunoprecipitants were analyzed by WB with anti-Flag, KIF7, SUFU and GLI2 antibodies. G P4HA2 interacts with GLI1. HEK293T cells were transfected with Flag-tagged P4HA2 and V5-tagged GLI1. Cell lysates were subjected to Flag IP. The immunoprecipitants were analyzed by WB with anti-Flag, KIF7 and V5 antibodies. H P4HA2 directly interacts with KIF7 in vitro. His-tagged KIF7, GLI1, SUFU, and Flag-tagged P4HA2 proteins were purified and incubated. After His pulldown, the complex was analyzed by WB. I KIF7 knockdown decreases the interaction between P4HA2 with SUFU. IgG-P4HA2 was overexpressed in shKIF7-1 and shKIF7-2 HEK293T cells. Cell lysates were subjected to IgG IP. Co-immunoprecipitated SUFU was detected by WB. J KIF7 knockdown decreases the interaction between P4HA2 and GLI1. IgG-P4HA2 and V5-GLI1 were co-overexpressed in KIF7 stable knockdown HEK293T cells (shKIF7-1 and shKIF7-2). Cell lysates were subjected to IgG IP. Co-immunoprecipitated V5-GLI1 was detected by WB.

Article Snippet: The following antibodies were used for western blotting (WB), immunofluorescence (IF): PAN-OH (home-made; 1:1,000 for WB); P4HA2 (13759-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC, 1:200 for IF); P4HA1 (12658-1-AP, rabbit, Proteintech; 1:1,000 for WB); KIF7 (24693-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC); SUFU (26759-1-AP, rabbit, Proteintech; 1:2,000 for WB); Flag (clone M2, F1804, mouse, Sigma-Aldrich; 1:10,000 for WB, 1:200 for IF); His (clone OGHis, D291-3, mouse, Medical & Biological Laboratories; 1:5,000 for WB); V5 (AB3792, rabbit, Merck Millipore; 1:2,000 for WB); GLI1 (clone A-7, sc-515781, mouse, Santa Cruz; 1:1,000 for WB, 1:200 for IHC); GLI2 (18989-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC) ; GAPDH (clone 1E6D9, 60004-1-Ig, mouse, Proteintech; 1:10,000 for WB); Arl13b (clone N295B/66, 75-287, mouse, NeuroMab; 1:200 for IF); Cy3 AffiniPure Goat Anti-Mouse IgG (H + L) (115-165-003, Jackson ImmunoResearch; 1:10,000 for IF); α-SMA (#34105, Cell Signaling Technology, 1:100 for IF) Light chain specific Mouse Anti-Rabbit IgG (211-032-171, Jackson ImmunoResearch; 1:10,000 for WB); Light chain specific Goat Anti Mouse IgG (115-035-174, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Rabbit IgG, Fc fragment specific (111-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Donkey anti-mouse IgG (H&L) (715-035-150, Jackson ImmunoResearch; 1:10,000 for WB); Goat Anti-Rabbit IgG (H + L) (111-035-003, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific (115-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Smoothened Agonist (SAG), Selleck, Cat# S7779; Fetal bovine serum, Thermo Fisher, Cat# 16140071; Penicillin/streptomycin, Thermo Fisher, Cat# 15140122; RPMI 1640, Thermo Fisher, Cat# 11875093; DMEM, Thermo Fisher, Cat# 11995065; Puromycin Dihydrochloride, Sigma, Cat# P8833; Trypsin 0.25% EDTA, Thermo Fisher, Cat# 25200072; Polyethylenimine Hydrochloride (MW 40,000), Polysciences, Cat# 24765-1; Polybrene Transfection Reagent, Sigma, Cat# TR-1003-G; SHH protein, Yun Zhao Laboratory; Anti-V5-tag mAb-Magnetic Beads, Medical & Biological Laboratories, Cat# M215-11; NTI-FLAG® M2 Affinity Gel, Sigma, Cat# F2426; Ni-NTA Agarose, QIAGEN, Cat# 30230; HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01/02; ChamQ SYBR qPCR Master Mix, Vazyme, Cat# Q311-02.

Techniques: Binding Assay, Activity Assay, Mutagenesis, Construct, Transfection, Immunoprecipitation, Western Blot, In Vitro, Purification, Incubation, Knockdown

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: A The trafficking of the P4HA2-KIF7 complex from the cytoplasm to the cilium responds to Hedgehog signaling. NIH/3T3 cells were co-infected with KIF7-BFP and P4HA2-EGFP lentivirus. White arrow indicates the co-localization of cilia, KIF7-BFP and P4HA2-EGFP. Yellow arrow indicates the co-localization of cilia and P4HA2-EGFP, which may bind with endogenous KIF7. Cells were treated with 200 nM SAG (+) or not (−). Cells were fixed and stained with antibodies for ARL13B to mark primary cilia. Scale bars: 5 μm. B Statistical analysis of the relative colocalization rate in ( A ) indicated cells. Data are shown as the mean ± SEM; SAG (−), n = 11; SAG (+), n = 13. ** P < 0.01. C The regulation of the Hedgehog signaling by P4ha2 is dependent on KIF7. KIF7 knockout (KO) and the control cells were knocked down with P4HA2 (shP4HA2). Cells were treated with (+) or without (−) 200 nM SAG. qRT-PCR analysis of GLI1 transcripts was performed using RNA isolated from the indicated cell lines. Data are shown as the mean ± SEM ( n = 3). *** P < 0.001. All experiments were repeated three times independently.

Article Snippet: The following antibodies were used for western blotting (WB), immunofluorescence (IF): PAN-OH (home-made; 1:1,000 for WB); P4HA2 (13759-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC, 1:200 for IF); P4HA1 (12658-1-AP, rabbit, Proteintech; 1:1,000 for WB); KIF7 (24693-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC); SUFU (26759-1-AP, rabbit, Proteintech; 1:2,000 for WB); Flag (clone M2, F1804, mouse, Sigma-Aldrich; 1:10,000 for WB, 1:200 for IF); His (clone OGHis, D291-3, mouse, Medical & Biological Laboratories; 1:5,000 for WB); V5 (AB3792, rabbit, Merck Millipore; 1:2,000 for WB); GLI1 (clone A-7, sc-515781, mouse, Santa Cruz; 1:1,000 for WB, 1:200 for IHC); GLI2 (18989-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC) ; GAPDH (clone 1E6D9, 60004-1-Ig, mouse, Proteintech; 1:10,000 for WB); Arl13b (clone N295B/66, 75-287, mouse, NeuroMab; 1:200 for IF); Cy3 AffiniPure Goat Anti-Mouse IgG (H + L) (115-165-003, Jackson ImmunoResearch; 1:10,000 for IF); α-SMA (#34105, Cell Signaling Technology, 1:100 for IF) Light chain specific Mouse Anti-Rabbit IgG (211-032-171, Jackson ImmunoResearch; 1:10,000 for WB); Light chain specific Goat Anti Mouse IgG (115-035-174, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Rabbit IgG, Fc fragment specific (111-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Donkey anti-mouse IgG (H&L) (715-035-150, Jackson ImmunoResearch; 1:10,000 for WB); Goat Anti-Rabbit IgG (H + L) (111-035-003, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific (115-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Smoothened Agonist (SAG), Selleck, Cat# S7779; Fetal bovine serum, Thermo Fisher, Cat# 16140071; Penicillin/streptomycin, Thermo Fisher, Cat# 15140122; RPMI 1640, Thermo Fisher, Cat# 11875093; DMEM, Thermo Fisher, Cat# 11995065; Puromycin Dihydrochloride, Sigma, Cat# P8833; Trypsin 0.25% EDTA, Thermo Fisher, Cat# 25200072; Polyethylenimine Hydrochloride (MW 40,000), Polysciences, Cat# 24765-1; Polybrene Transfection Reagent, Sigma, Cat# TR-1003-G; SHH protein, Yun Zhao Laboratory; Anti-V5-tag mAb-Magnetic Beads, Medical & Biological Laboratories, Cat# M215-11; NTI-FLAG® M2 Affinity Gel, Sigma, Cat# F2426; Ni-NTA Agarose, QIAGEN, Cat# 30230; HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01/02; ChamQ SYBR qPCR Master Mix, Vazyme, Cat# Q311-02.

Techniques: Infection, Staining, Knock-Out, Control, Quantitative RT-PCR, Isolation

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: A , B The regulation of Hh signaling by P4HA2 is dependent on P4HA2 hydroxylase activity. HEK293T P4HA2 KO cells were expressed with wild-type P4HA2 (P4HA2) or hydroxylase dominant negative mutant (P4HA2-HDH). Cells were treated with (+) or without (−) 200 nM SAG. qRT-PCR analysis of GLI1 ( A ) and GLI2 ( B ) transcripts was performed using RNA isolated from the indicated cell lines. Data are shown as the mean ± SEM ( n = 3). * P < 0.05, *** P < 0.001. All experiments were repeated three times independently. C – E The hydroxylation levels of GLI1 and SUFU decrease in P4HA2 knockout cells. P4HA2 KO and the vehicle cells were transfected with Flag-tagged KIF7, GLI1, and SUFU. Cell lysates were subjected to Flag IP and then analyzed by WB. Hydroxylated proteins were recognized by the Pan-hydroxylation antibody (OH). The relative quantification of Flag-KIF7 ( C ), GLI1 ( D ), and SUFU ( E ) hydroxylation level was analyzed and normalized. The values shown are the means of three independent experiments. F – H LC-MS/MS spectra of tryptic peptides. 3 tryptic peptides Pro 46 ( F ), Pro 134 ( G ), and Pro 249 ( H ) from SUFU. The b ion and y ion are fragment ions of tryptic peptide in tandem mass spectrometry. The x and y axes represent m/z and relative ion intensity, respectively. I Validation of the proline 46, proline 134, and proline 249 hydroxylation sites of SUFU. P46A, P134A, P249A, and P46/134/249A proline to alanine mutants of SUFU were constructed and transfected into HEK293T cells. Cell lysates were subjected to Flag IP. The relative quantification of SUFU WT and mutant hydroxylation levels was analyzed and normalized. The values shown are the means of three independent experiments. J P4HA2 regulates the protein stability of SUFU in response to the Hh pathway activation. HEK293T P4HA2 KO cells were expressed with wild-type P4HA2 or a P4HA2 dominant negative mutant in hydroxylase activity (HDH). Cell lysates were analyzed by WB with anti-P4HA2 and GAPDH antibodies. K Hydroxylated SUFU regulate the Hh signaling. SUFU knockout (KO) NIH/3T3 and control cell lines were constructed and then infected with SUFU WT and mutant (SUFU P46/134/627 A) lentivirus. qRT-PCR analysis of GLI1 transcripts was performed using RNA isolated from the above cell lines. L SUFU KO NIH/3T3 and control cell lines were constructed and then infected with SUFU WT and mutant (SUFU P46/134/627 A) lentivirus. Utilized a dual luciferase reporter gene system, GLI1-Firefly and Renilla plasmids were overexpressed in above cell lines. Relative fold change of GLI1 luciferase activity was measured and normalized. Data are shown as the mean ± SEM ( n = 3). *** P < 0.001, **** P < 0.0001. All experiments were repeated three times independently.

Article Snippet: The following antibodies were used for western blotting (WB), immunofluorescence (IF): PAN-OH (home-made; 1:1,000 for WB); P4HA2 (13759-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC, 1:200 for IF); P4HA1 (12658-1-AP, rabbit, Proteintech; 1:1,000 for WB); KIF7 (24693-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC); SUFU (26759-1-AP, rabbit, Proteintech; 1:2,000 for WB); Flag (clone M2, F1804, mouse, Sigma-Aldrich; 1:10,000 for WB, 1:200 for IF); His (clone OGHis, D291-3, mouse, Medical & Biological Laboratories; 1:5,000 for WB); V5 (AB3792, rabbit, Merck Millipore; 1:2,000 for WB); GLI1 (clone A-7, sc-515781, mouse, Santa Cruz; 1:1,000 for WB, 1:200 for IHC); GLI2 (18989-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC) ; GAPDH (clone 1E6D9, 60004-1-Ig, mouse, Proteintech; 1:10,000 for WB); Arl13b (clone N295B/66, 75-287, mouse, NeuroMab; 1:200 for IF); Cy3 AffiniPure Goat Anti-Mouse IgG (H + L) (115-165-003, Jackson ImmunoResearch; 1:10,000 for IF); α-SMA (#34105, Cell Signaling Technology, 1:100 for IF) Light chain specific Mouse Anti-Rabbit IgG (211-032-171, Jackson ImmunoResearch; 1:10,000 for WB); Light chain specific Goat Anti Mouse IgG (115-035-174, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Rabbit IgG, Fc fragment specific (111-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Donkey anti-mouse IgG (H&L) (715-035-150, Jackson ImmunoResearch; 1:10,000 for WB); Goat Anti-Rabbit IgG (H + L) (111-035-003, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific (115-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Smoothened Agonist (SAG), Selleck, Cat# S7779; Fetal bovine serum, Thermo Fisher, Cat# 16140071; Penicillin/streptomycin, Thermo Fisher, Cat# 15140122; RPMI 1640, Thermo Fisher, Cat# 11875093; DMEM, Thermo Fisher, Cat# 11995065; Puromycin Dihydrochloride, Sigma, Cat# P8833; Trypsin 0.25% EDTA, Thermo Fisher, Cat# 25200072; Polyethylenimine Hydrochloride (MW 40,000), Polysciences, Cat# 24765-1; Polybrene Transfection Reagent, Sigma, Cat# TR-1003-G; SHH protein, Yun Zhao Laboratory; Anti-V5-tag mAb-Magnetic Beads, Medical & Biological Laboratories, Cat# M215-11; NTI-FLAG® M2 Affinity Gel, Sigma, Cat# F2426; Ni-NTA Agarose, QIAGEN, Cat# 30230; HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01/02; ChamQ SYBR qPCR Master Mix, Vazyme, Cat# Q311-02.

Techniques: Activity Assay, Dominant Negative Mutation, Quantitative RT-PCR, Isolation, Knock-Out, Transfection, Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Biomarker Discovery, Construct, Mutagenesis, Activation Assay, Control, Infection, Luciferase

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: A – D Injection of luciferased Eµ-myc arf −/ − B cells into C57BL/6 WT and P4ha2 −/ − mice. Bioluminescence imaging ( A , C ) of mice for 18 days after injection. Luminescence intensity curves ( B, D ) for mice injected with luciferased Eµ-myc arf −/ − B cells. E P4HA2 is located in stromal fibroblasts. Immunofluorescence staining of α-SMA, P4HA2 and DAPI in C57BL/6 WT mice xenograft. Scale bars: 100 μm. F Primary stromal fibroblasts were isolated from bone marrow of C57BL/6 WT and P4ha2 −/ − mice. qRT-PCR analysis of Gli2, Ptch1, Ptch2 and HH ligands transcripts was performed using RNA isolated from the primary stromal fibroblasts. G Differential genes of Hh downstream secreted factors obtained from RNA-seq analysis of primary bone marrow stromal fibroblasts. Data are shown as the mean ± SEM ( n = 3). * P < 0.1, ** P < 0.01, *** P < 0.001. H The growth curve of Eµ-myc arf −/ − B cells co-cultured with primary bone marrow stromal fibroblasts supernatant. WT and P4ha2 −/ − primary bone marrow fibroblasts were isolated and cultured. After cell counting, both primary cells were cultured at the same density, and the cultured supernatant was collected for co-culture with Eµ-myc arf −/ − B cells. The cell viability was detected every 12 hr. I qRT-PCR analysis of Gli1 and Gli2 transcripts was performed using the tissue of C57BL/6 WT and P4ha2 −/ − mice xenograft. Data are shown as the mean ± SEM ( n = 3). *** P < 0.001, **** P < 0.0001. All experiments were repeated three times independently.

Article Snippet: The following antibodies were used for western blotting (WB), immunofluorescence (IF): PAN-OH (home-made; 1:1,000 for WB); P4HA2 (13759-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC, 1:200 for IF); P4HA1 (12658-1-AP, rabbit, Proteintech; 1:1,000 for WB); KIF7 (24693-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC); SUFU (26759-1-AP, rabbit, Proteintech; 1:2,000 for WB); Flag (clone M2, F1804, mouse, Sigma-Aldrich; 1:10,000 for WB, 1:200 for IF); His (clone OGHis, D291-3, mouse, Medical & Biological Laboratories; 1:5,000 for WB); V5 (AB3792, rabbit, Merck Millipore; 1:2,000 for WB); GLI1 (clone A-7, sc-515781, mouse, Santa Cruz; 1:1,000 for WB, 1:200 for IHC); GLI2 (18989-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC) ; GAPDH (clone 1E6D9, 60004-1-Ig, mouse, Proteintech; 1:10,000 for WB); Arl13b (clone N295B/66, 75-287, mouse, NeuroMab; 1:200 for IF); Cy3 AffiniPure Goat Anti-Mouse IgG (H + L) (115-165-003, Jackson ImmunoResearch; 1:10,000 for IF); α-SMA (#34105, Cell Signaling Technology, 1:100 for IF) Light chain specific Mouse Anti-Rabbit IgG (211-032-171, Jackson ImmunoResearch; 1:10,000 for WB); Light chain specific Goat Anti Mouse IgG (115-035-174, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Rabbit IgG, Fc fragment specific (111-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Donkey anti-mouse IgG (H&L) (715-035-150, Jackson ImmunoResearch; 1:10,000 for WB); Goat Anti-Rabbit IgG (H + L) (111-035-003, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific (115-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Smoothened Agonist (SAG), Selleck, Cat# S7779; Fetal bovine serum, Thermo Fisher, Cat# 16140071; Penicillin/streptomycin, Thermo Fisher, Cat# 15140122; RPMI 1640, Thermo Fisher, Cat# 11875093; DMEM, Thermo Fisher, Cat# 11995065; Puromycin Dihydrochloride, Sigma, Cat# P8833; Trypsin 0.25% EDTA, Thermo Fisher, Cat# 25200072; Polyethylenimine Hydrochloride (MW 40,000), Polysciences, Cat# 24765-1; Polybrene Transfection Reagent, Sigma, Cat# TR-1003-G; SHH protein, Yun Zhao Laboratory; Anti-V5-tag mAb-Magnetic Beads, Medical & Biological Laboratories, Cat# M215-11; NTI-FLAG® M2 Affinity Gel, Sigma, Cat# F2426; Ni-NTA Agarose, QIAGEN, Cat# 30230; HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01/02; ChamQ SYBR qPCR Master Mix, Vazyme, Cat# Q311-02.

Techniques: Injection, Imaging, Immunofluorescence, Staining, Isolation, Quantitative RT-PCR, RNA Sequencing, Cell Culture, Cell Counting, Co-Culture 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: 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" t5 caption a7 Mutant RANTES Response (CCR5 DiscoveRx) IC 50 Overall Rank CHO-CCR2 xCelligence CCR5 DiscoveRx CCR2 DiscoveRx CCR7 DiscoveRx MΦ xCelligence % nM WT 35.1 90.0 6.6 ± 0.9 (3) 13.9 ± 1 (3) 12.2 ± 0.4 (2) 3 ± 0.8 (3) 3 Y80A 95.6 >1000 10 R89A 15.1 20.6 3.7 ± 1.7 (4) * 3.1 ± 0.4 (2) *** 4.1 ± 0.1 (2) ** 1 ± 0.4 (3) * 1 D141A 74.3 139.0 4 D141L 80.6 >750 8 D141R 79.4 >700 6 E143A 71.7 5 E143K 93.9 >5000 N.D. (3) N.D. (2) N.D. (3) 11 E143R 92.5 11 V185A 26.5 50.0 2 Y217A 66.5 >5000 7 Y217N 67.5 >1400 6 MR-Fc 91.2 >1400 N.D. (3) N.D. (2) N.D. (3) 9 Open in a separate window N.D., IC 50 could not be determined (i.e., a curve could not be fitted).

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" t5 caption a7 Mutant RANTES Response (CCR5 DiscoveRx) IC 50 Overall Rank CHO-CCR2 xCelligence CCR5 DiscoveRx CCR2 DiscoveRx CCR7 DiscoveRx MΦ xCelligence % nM WT 35.1 90.0 6.6 ± 0.9 (3) 13.9 ± 1 (3) 12.2 ± 0.4 (2) 3 ± 0.8 (3) 3 Y80A 95.6 >1000 10 R89A 15.1 20.6 3.7 ± 1.7 (4) * 3.1 ± 0.4 (2) *** 4.1 ± 0.1 (2) ** 1 ± 0.4 (3) * 1 D141A 74.3 139.0 4 D141L 80.6 >750 8 D141R 79.4 >700 6 E143A 71.7 5 E143K 93.9 >5000 N.D. (3) N.D. (2) N.D. (3) 11 E143R 92.5 11 V185A 26.5 50.0 2 Y217A 66.5 >5000 7 Y217N 67.5 >1400 6 MR-Fc 91.2 >1400 N.D. (3) N.D. (2) N.D. (3) 9 Open in a separate window N.D., IC 50 could not be determined (i.e., a curve could not be fitted).

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" t5 caption a7 Mutant RANTES Response (CCR5 DiscoveRx) IC 50 Overall Rank CHO-CCR2 xCelligence CCR5 DiscoveRx CCR2 DiscoveRx CCR7 DiscoveRx MΦ xCelligence % nM WT 35.1 90.0 6.6 ± 0.9 (3) 13.9 ± 1 (3) 12.2 ± 0.4 (2) 3 ± 0.8 (3) 3 Y80A 95.6 >1000 10 R89A 15.1 20.6 3.7 ± 1.7 (4) * 3.1 ± 0.4 (2) *** 4.1 ± 0.1 (2) ** 1 ± 0.4 (3) * 1 D141A 74.3 139.0 4 D141L 80.6 >750 8 D141R 79.4 >700 6 E143A 71.7 5 E143K 93.9 >5000 N.D. (3) N.D. (2) N.D. (3) 11 E143R 92.5 11 V185A 26.5 50.0 2 Y217A 66.5 >5000 7 Y217N 67.5 >1400 6 MR-Fc 91.2 >1400 N.D. (3) N.D. (2) N.D. (3) 9 Open in a separate window N.D., IC 50 could not be determined (i.e., a curve could not be fitted).

Techniques: Beta-Arrestin Assay, Activation Assay, Concentration Assay