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htlr5-ptgfp1  (Lonza)


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    Structured Review

    Lonza htlr5-ptgfp1
    The activity of bovine and <t>human</t> <t>TLR5</t> differs with cell background. CXCL8 protein release from ( a ) HEK293T cells and ( b ) EBL cells stably transfected with human TLR5 (♦) or bovine TLR5 (■), activated with E. coli H7 flagellin at various concentrations. The Figures show the average CXCL8 protein response for each H7 concentration as a percentage of the background response in non-transfected cells. The error bars illustrate the standard error of the mean for three experiments, each with three technical replicates. The average CXCL8 protein release in response to flagellin was significantly different in cells expressing bovine or human forms of TLR5 by ANOVA (P < 0.001) and * denotes which doses were significant by subsequent Tukey’s test (P < 0.05).
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    1) Product Images from "Host species adaptation of TLR5 signalling and flagellin recognition"

    Article Title: Host species adaptation of TLR5 signalling and flagellin recognition

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17935-5

    The activity of bovine and human TLR5 differs with cell background. CXCL8 protein release from ( a ) HEK293T cells and ( b ) EBL cells stably transfected with human TLR5 (♦) or bovine TLR5 (■), activated with E. coli H7 flagellin at various concentrations. The Figures show the average CXCL8 protein response for each H7 concentration as a percentage of the background response in non-transfected cells. The error bars illustrate the standard error of the mean for three experiments, each with three technical replicates. The average CXCL8 protein release in response to flagellin was significantly different in cells expressing bovine or human forms of TLR5 by ANOVA (P < 0.001) and * denotes which doses were significant by subsequent Tukey’s test (P < 0.05).
    Figure Legend Snippet: The activity of bovine and human TLR5 differs with cell background. CXCL8 protein release from ( a ) HEK293T cells and ( b ) EBL cells stably transfected with human TLR5 (♦) or bovine TLR5 (■), activated with E. coli H7 flagellin at various concentrations. The Figures show the average CXCL8 protein response for each H7 concentration as a percentage of the background response in non-transfected cells. The error bars illustrate the standard error of the mean for three experiments, each with three technical replicates. The average CXCL8 protein release in response to flagellin was significantly different in cells expressing bovine or human forms of TLR5 by ANOVA (P < 0.001) and * denotes which doses were significant by subsequent Tukey’s test (P < 0.05).

    Techniques Used: Activity Assay, Stable Transfection, Transfection, Concentration Assay, Expressing

    Blocking TLR signalling with down-stream inhibitors and targeted siRNA reveals similarities and differences between human and bovine signalling pathways. HEK293T cells stably transfected with human TLR5 ( a ) and EBL cells stably transfected with bovine TLR5 ( c ) were transiently transfected with siRNA for p38, RELA, TRAF6 and PIK3R1. PIK3R1 (human) and PIK3R1#b3 (bovine) are predicted to recognize all splice variants, thereby reducing levels of all three regulatory subunits of class IA PI3K encoded by human or bovine PIK3R1 respectively; PIK3R1#b2 (bovine) is predicted to only recognize the splice variant encoding the p85A regulatory subunit. In addition, cells were treated with transfection reagent only (TC) or transfected with non-target control siRNA (NTC). After 24hr cells were activated with 100 ng/ml E. coli H7 flagellin. The graphs illustrates the average release of CXCL8 protein relative to that released by NTC samples. The error bars illustrate the standard error of the mean of three experiments, each with three technical replicates. HEK293T cells stably transfected with human TLR5 ( b ) and EBL cells stably transfected with bovine TLR5 ( d ) were treated with inhibitors of p38 (5 μM SB203580), NF-κB (100 μM PDTC) and PI3K (50 μM LY294002) or DMSO before stimulation with ng/ml E. coli H7 flagellin. The graphs illustrates the average release of CXCL8 protein relative to that released by DMSO treated samples. The error bars illustrate the standard error of the mean of three experiments, each with three technical replicates. *Denotes that the average CXCL8 level was statistically significantly different from ( a and c ) NTC or ( b and d ) DMSO and H7 treated samples by ANOVA and subsequent Tukey’s test (P < 0.05).
    Figure Legend Snippet: Blocking TLR signalling with down-stream inhibitors and targeted siRNA reveals similarities and differences between human and bovine signalling pathways. HEK293T cells stably transfected with human TLR5 ( a ) and EBL cells stably transfected with bovine TLR5 ( c ) were transiently transfected with siRNA for p38, RELA, TRAF6 and PIK3R1. PIK3R1 (human) and PIK3R1#b3 (bovine) are predicted to recognize all splice variants, thereby reducing levels of all three regulatory subunits of class IA PI3K encoded by human or bovine PIK3R1 respectively; PIK3R1#b2 (bovine) is predicted to only recognize the splice variant encoding the p85A regulatory subunit. In addition, cells were treated with transfection reagent only (TC) or transfected with non-target control siRNA (NTC). After 24hr cells were activated with 100 ng/ml E. coli H7 flagellin. The graphs illustrates the average release of CXCL8 protein relative to that released by NTC samples. The error bars illustrate the standard error of the mean of three experiments, each with three technical replicates. HEK293T cells stably transfected with human TLR5 ( b ) and EBL cells stably transfected with bovine TLR5 ( d ) were treated with inhibitors of p38 (5 μM SB203580), NF-κB (100 μM PDTC) and PI3K (50 μM LY294002) or DMSO before stimulation with ng/ml E. coli H7 flagellin. The graphs illustrates the average release of CXCL8 protein relative to that released by DMSO treated samples. The error bars illustrate the standard error of the mean of three experiments, each with three technical replicates. *Denotes that the average CXCL8 level was statistically significantly different from ( a and c ) NTC or ( b and d ) DMSO and H7 treated samples by ANOVA and subsequent Tukey’s test (P < 0.05).

    Techniques Used: Blocking Assay, Stable Transfection, Transfection, Variant Assay

    Diverse flagellin alignments of Nt and Ct D1 domains, indicating identified TLR5 binding amino acids and relative responses to these by bovine and human TLR5. ( a ) 1 Numbering according to Smith et al . (2003) . 2 Blue amino acids bind to TLR5 binding interface-B; green amino-acids bind to TLR5 binding interface-A, according to Yoon et al . (2012) ; Black amino acids bind to TLR5 according to Smith et al . (2003) . aas: amino acids. ( b ) The relative CXCL8 protein release from HEK293T cells stably transfected with human TLR5 and EBL cells stably transfected with bovine TLR5 after stimulation with 50ng/ml S . Typhimurium P1 (St P1), Listeria ivanovii FlaA (Li FlaA) and Burkholderia thailandensis FliC (Bt FliC) compared to the response to E. coli H7 (Ec H7). Error bars illustrate the standard errors of the mean for three experiments, each with three technical replicates. *Denotes that the average CXCL8 protein release in response to the different flagellin was significantly different by ANOVA and subsequent Tukey’s test (P < 0.05).
    Figure Legend Snippet: Diverse flagellin alignments of Nt and Ct D1 domains, indicating identified TLR5 binding amino acids and relative responses to these by bovine and human TLR5. ( a ) 1 Numbering according to Smith et al . (2003) . 2 Blue amino acids bind to TLR5 binding interface-B; green amino-acids bind to TLR5 binding interface-A, according to Yoon et al . (2012) ; Black amino acids bind to TLR5 according to Smith et al . (2003) . aas: amino acids. ( b ) The relative CXCL8 protein release from HEK293T cells stably transfected with human TLR5 and EBL cells stably transfected with bovine TLR5 after stimulation with 50ng/ml S . Typhimurium P1 (St P1), Listeria ivanovii FlaA (Li FlaA) and Burkholderia thailandensis FliC (Bt FliC) compared to the response to E. coli H7 (Ec H7). Error bars illustrate the standard errors of the mean for three experiments, each with three technical replicates. *Denotes that the average CXCL8 protein release in response to the different flagellin was significantly different by ANOVA and subsequent Tukey’s test (P < 0.05).

    Techniques Used: Binding Assay, Stable Transfection, Transfection

    Related Articles

    Transfection:

    Article Title: Host species adaptation of TLR5 signalling and flagellin recognition
    Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a NucleofectorTM 2d Device (Lonza Group Ltd., US) as detailed previously .

    Electroporation:

    Article Title: Host species adaptation of TLR5 signalling and flagellin recognition
    Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a NucleofectorTM 2d Device (Lonza Group Ltd., US) as detailed previously .

    Construct:

    Article Title: Host species adaptation of TLR5 signalling and flagellin recognition
    Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a NucleofectorTM 2d Device (Lonza Group Ltd., US) as detailed previously .

    Plasmid Preparation:

    Article Title: Host species adaptation of TLR5 signalling and flagellin recognition
    Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a NucleofectorTM 2d Device (Lonza Group Ltd., US) as detailed previously .

    Activity Assay:

    Article Title: Host species adaptation of TLR5 signalling and flagellin recognition
    Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a NucleofectorTM 2d Device (Lonza Group Ltd., US) as detailed previously .

    Stable Transfection:

    Article Title: Host species adaptation of TLR5 signalling and flagellin recognition
    Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a NucleofectorTM 2d Device (Lonza Group Ltd., US) as detailed previously .

    Concentration Assay:

    Article Title: Host species adaptation of TLR5 signalling and flagellin recognition
    Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a NucleofectorTM 2d Device (Lonza Group Ltd., US) as detailed previously .

    Expressing:

    Article Title: Host species adaptation of TLR5 signalling and flagellin recognition
    Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a NucleofectorTM 2d Device (Lonza Group Ltd., US) as detailed previously .

    Blocking Assay:

    Article Title: Host species adaptation of TLR5 signalling and flagellin recognition
    Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a NucleofectorTM 2d Device (Lonza Group Ltd., US) as detailed previously .

    Variant Assay:

    Article Title: Host species adaptation of TLR5 signalling and flagellin recognition
    Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a NucleofectorTM 2d Device (Lonza Group Ltd., US) as detailed previously .

    Binding Assay:

    Article Title: Host species adaptation of TLR5 signalling and flagellin recognition
    Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a NucleofectorTM 2d Device (Lonza Group Ltd., US) as detailed previously .

    Mutagenesis:

    Article Title: Host species adaptation of TLR5 signalling and flagellin recognition
    Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a NucleofectorTM 2d Device (Lonza Group Ltd., US) as detailed previously .



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    a, HCT116 cells stably expressing BRIP1 R162Q were transiently transfected using PEI with empty vector (EV-EGFP) or RNaseH1-WT-eGFP. Transfection efficiency was assessed via EGFP fluorescence. b, immunoblot analysis confirming expression of GFP-tagged constructs in transfected cells. c, Immunofluorescence detection of R-loops using the S9.6 antibody in cells transfected with EGFP or EGFP-RNaseH1. d, Quantification of S9.6 mean fluorescence intensity (MFI) per nucleus. Data represent mean ± SEM of three independent experiments. e, Immunofluorescence detection of G-quadruplex (G4) structures using the BG4 antibody in cells transfected with EGFP or EGFP-RNaseH1. f, Quantification of BG4 fluorescence intensity per nucleus. Data represent mean ± SEM of three independent experiments. g, Representative DNA fiber assay images from HCT116 cells harboring BRIP1 R162Q transfected with empty vector (EV-eGFP) or RNaseH1-WT. DNA replication tracts were sequentially labeled with CldU (red) and IdU (green). h, Quantification of DNA fiber types. RNaseH1-WT expression reduced the proportion of stalled replication forks in cells harboring BRIP1 R162Q. i, While the overall fork speed was not significantly increased upon RNaseH1-WT expression, tracts displayed a more balanced distribution of CldU and IdU incorporation. j, Fork asymmetry analysis. RNaseH1-WT expression reduced fork asymmetry, indicating that left- and right-moving sister forks progressed at more similar speeds. Labeled tracks were visualized by confocal microscopy, and tract lengths were measured on raw LSM images. Lengths (µm) were converted to replication speed (kb/min) using a factor of 2.59 kb/µm. All data represent mean ± SEM from three independent experiments. At least 150 fibers per condition were analyzed in each replicate. Statistical significance was determined using paired two-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant). c, e, Representative confocal images are shown.

    Journal: bioRxiv

    Article Title: Role of a childhood cancer-linked BRIP1/FANCJ germline variant in genomic instability and cancer cell vulnerability

    doi: 10.64898/2026.03.24.714005

    Figure Lengend Snippet: a, HCT116 cells stably expressing BRIP1 R162Q were transiently transfected using PEI with empty vector (EV-EGFP) or RNaseH1-WT-eGFP. Transfection efficiency was assessed via EGFP fluorescence. b, immunoblot analysis confirming expression of GFP-tagged constructs in transfected cells. c, Immunofluorescence detection of R-loops using the S9.6 antibody in cells transfected with EGFP or EGFP-RNaseH1. d, Quantification of S9.6 mean fluorescence intensity (MFI) per nucleus. Data represent mean ± SEM of three independent experiments. e, Immunofluorescence detection of G-quadruplex (G4) structures using the BG4 antibody in cells transfected with EGFP or EGFP-RNaseH1. f, Quantification of BG4 fluorescence intensity per nucleus. Data represent mean ± SEM of three independent experiments. g, Representative DNA fiber assay images from HCT116 cells harboring BRIP1 R162Q transfected with empty vector (EV-eGFP) or RNaseH1-WT. DNA replication tracts were sequentially labeled with CldU (red) and IdU (green). h, Quantification of DNA fiber types. RNaseH1-WT expression reduced the proportion of stalled replication forks in cells harboring BRIP1 R162Q. i, While the overall fork speed was not significantly increased upon RNaseH1-WT expression, tracts displayed a more balanced distribution of CldU and IdU incorporation. j, Fork asymmetry analysis. RNaseH1-WT expression reduced fork asymmetry, indicating that left- and right-moving sister forks progressed at more similar speeds. Labeled tracks were visualized by confocal microscopy, and tract lengths were measured on raw LSM images. Lengths (µm) were converted to replication speed (kb/min) using a factor of 2.59 kb/µm. All data represent mean ± SEM from three independent experiments. At least 150 fibers per condition were analyzed in each replicate. Statistical significance was determined using paired two-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant). c, e, Representative confocal images are shown.

    Article Snippet: For R-loop removal, cells were transiently transfected with an EGFP empty vector control (pEGFP-C1, Takara Bio, #632470), EGFP-tagged RNaseH1 wild-type (WT), or the catalytically inactive RNaseH1 D145N mutant as indicated.

    Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Fluorescence, Western Blot, Construct, Immunofluorescence, Labeling, Confocal Microscopy, Two Tailed Test

    Overexpression of SNHG1 or HDAC6 partially reverses the pro-angiogenic effect of EPC-EVs on MAECs. MAECs were transfected with SNHG1 overexpression vectors (oe-SNHG1), HDAC6 overexpression vectors (oe-HDAC6), or empty vectors (oe-NC, control) in HG (30 mM) medium. A : Transfection efficiency of oe-SNHG1 and oe-HDAC6 was verified by qRT-PCR (n = 3). Cells were then co-treated with EPC-EVs-miR in HG medium. B : SNHG1 and HDAC6 expression was detected by qRT-PCR (n = 3). C : Expression of HDAC6, CD31, and VEGFA was measured by Western blot (n = 3). D : Cell viability was assessed by CCK-8 assay (n = 3). E : Angiogenic capacity was evaluated by tube formation assay (n = 3). F : Cell migration was measured by scratch wound healing assay (n = 3). Data are presented as mean ± standard deviation. Data in panel A were analyze using student’s t-test. Data in panels B , D , E , and F were analyzed using one-way ANOVA. Data in panel C were analyzed using two-way ANOVA, followed by Tukey’s multiple comparisons test. * p < 0.05 and ** p < 0.01

    Journal: Cell Biology and Toxicology

    Article Title: Endothelial progenitor cell derived extracellular vesicles promotes wound healing in diabetic mice via activating mobilization and neovascularization

    doi: 10.1007/s10565-025-10134-3

    Figure Lengend Snippet: Overexpression of SNHG1 or HDAC6 partially reverses the pro-angiogenic effect of EPC-EVs on MAECs. MAECs were transfected with SNHG1 overexpression vectors (oe-SNHG1), HDAC6 overexpression vectors (oe-HDAC6), or empty vectors (oe-NC, control) in HG (30 mM) medium. A : Transfection efficiency of oe-SNHG1 and oe-HDAC6 was verified by qRT-PCR (n = 3). Cells were then co-treated with EPC-EVs-miR in HG medium. B : SNHG1 and HDAC6 expression was detected by qRT-PCR (n = 3). C : Expression of HDAC6, CD31, and VEGFA was measured by Western blot (n = 3). D : Cell viability was assessed by CCK-8 assay (n = 3). E : Angiogenic capacity was evaluated by tube formation assay (n = 3). F : Cell migration was measured by scratch wound healing assay (n = 3). Data are presented as mean ± standard deviation. Data in panel A were analyze using student’s t-test. Data in panels B , D , E , and F were analyzed using one-way ANOVA. Data in panel C were analyzed using two-way ANOVA, followed by Tukey’s multiple comparisons test. * p < 0.05 and ** p < 0.01

    Article Snippet: For lentiviral infection, GFP-labeled miR-204-5p lentiviral overexpression vectors, SNHG1 lentiviral overexpression vectors, or control empty vectors (NC) (Genechem, Shanghai, China) were applied to transfection with 293 T cells (CRL-1573; ATCC, Manassas, VA, USA).

    Techniques: Over Expression, Transfection, Control, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay, Tube Formation Assay, Migration, Wound Healing Assay, Standard Deviation