strain towne  (ATCC)


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    Structured Review

    ATCC strain towne
    Strain Towne, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hcmv strains towne  (ATCC)


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    Structured Review

    ATCC hcmv strains towne
    Human cytomegalovirus <t>(HCMV)</t> prevents AKT activation in response to serum. (A) Stimulation of various receptor tyrosine kinases (RTKs) and other growth factor receptors causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, activating its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family transcription factors, and the tuberous sclerosis complex 2 (TSC2) protei. Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. (Schematic generated in BioRender.) (B) Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, and transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared with AKT (pan) antibody. (C–G) Fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain <t>Towne</t> at multiplicity of infection (MOI) of 2 (MOI = 2 TCID50/cell) for the indicated times, hours post-infection (hpi). Cells were then either treated (or mock treated) with serum for 10 min prior to lysis for Western blot analysis. (E–G) Fluorescent signals from dye-labeled secondary antibodies were quantified, and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473, and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used to visualize protein loading and confirm efficient transfer.
    Hcmv Strains Towne, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hcmv strains towne/product/ATCC
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hcmv strains towne - by Bioz Stars, 2023-11
    86/100 stars

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    1) Product Images from "Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins"

    Article Title: Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins

    Journal: Journal of Virology

    doi: 10.1128/jvi.00563-23

    Human cytomegalovirus (HCMV) prevents AKT activation in response to serum. (A) Stimulation of various receptor tyrosine kinases (RTKs) and other growth factor receptors causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, activating its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family transcription factors, and the tuberous sclerosis complex 2 (TSC2) protei. Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. (Schematic generated in BioRender.) (B) Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, and transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared with AKT (pan) antibody. (C–G) Fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne at multiplicity of infection (MOI) of 2 (MOI = 2 TCID50/cell) for the indicated times, hours post-infection (hpi). Cells were then either treated (or mock treated) with serum for 10 min prior to lysis for Western blot analysis. (E–G) Fluorescent signals from dye-labeled secondary antibodies were quantified, and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473, and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used to visualize protein loading and confirm efficient transfer.
    Figure Legend Snippet: Human cytomegalovirus (HCMV) prevents AKT activation in response to serum. (A) Stimulation of various receptor tyrosine kinases (RTKs) and other growth factor receptors causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, activating its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family transcription factors, and the tuberous sclerosis complex 2 (TSC2) protei. Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. (Schematic generated in BioRender.) (B) Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, and transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared with AKT (pan) antibody. (C–G) Fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne at multiplicity of infection (MOI) of 2 (MOI = 2 TCID50/cell) for the indicated times, hours post-infection (hpi). Cells were then either treated (or mock treated) with serum for 10 min prior to lysis for Western blot analysis. (E–G) Fluorescent signals from dye-labeled secondary antibodies were quantified, and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473, and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used to visualize protein loading and confirm efficient transfer.

    Techniques Used: Activation Assay, Scaffolding, Activity Assay, Generated, Lysis, SDS Page, Western Blot, Infection, Labeling, Staining

    Membrane recruitment of AKT is defective in HCMV-infected cells. (A–B) Serum-starved fibroblasts were either mock infected or infected with HCMV strain Towne (MOI= 2 TCID50/cell) for 12 h and either mock treated or treated with 5% newborn calf serum (NCS) for 10 min. Samples were mechanically lysed and differentially centrifuged to separate the membrane (M) fraction from the cytoplasmic (C) fraction and subjugated to Western blot analysis of indicated proteins alongside total (T) lysate. Ponceau staining was used as a loading control. (B) Detection signals from AKT (pan) bands were quantified for each condition, and bands from membrane fractions were normalized to signal cognate “total lysate” bands. Results were then normalized to the “No Serum” condition, the arithmetic mean was calculated, and values were graphed. A one-way ANOVA was run using Tukey’s multiple comparison post-test for statistical analysis (*P < 0.05, ****P < 0.0001). Error bars indicate standard error of the mean (SEM). n = 4 independent biological replicates.
    Figure Legend Snippet: Membrane recruitment of AKT is defective in HCMV-infected cells. (A–B) Serum-starved fibroblasts were either mock infected or infected with HCMV strain Towne (MOI= 2 TCID50/cell) for 12 h and either mock treated or treated with 5% newborn calf serum (NCS) for 10 min. Samples were mechanically lysed and differentially centrifuged to separate the membrane (M) fraction from the cytoplasmic (C) fraction and subjugated to Western blot analysis of indicated proteins alongside total (T) lysate. Ponceau staining was used as a loading control. (B) Detection signals from AKT (pan) bands were quantified for each condition, and bands from membrane fractions were normalized to signal cognate “total lysate” bands. Results were then normalized to the “No Serum” condition, the arithmetic mean was calculated, and values were graphed. A one-way ANOVA was run using Tukey’s multiple comparison post-test for statistical analysis (*P < 0.05, ****P < 0.0001). Error bars indicate standard error of the mean (SEM). n = 4 independent biological replicates.

    Techniques Used: Membrane, Infection, Western Blot, Staining, Comparison

    UV-treated virus fails to inactivate AKT. (A) HCMV strain Towne was mock treated or treated with 125 mJ of UV light for indicated seconds (sec) and then used to infect fibroblasts (MOI = 2 TCID50/cell) for 12 h. A Western blot was performed to monitor for IE1 expression, using Ponceau S stain to indicate total protein loading. n = 1. (B–C) Fibroblasts were infected with mock-treated or UV-treated HCMV strain Towne virus (MOI = 2 TCID50/cell) in the presence of serum, and lysates were taken at the indicated times post-infection (in hours), hours post-infection (hpi). A Western blot was performed probing for the indicated proteins; additionally, Ponceau S staining is shown as a readout for total protein loading. (C) Detection signals from 24 hpi bands were quantified, normalized to signals from 1 hpi bands, and the arithmetic means from three independent biological replicates were plotted. A one-way ANOVA with Tukey’s multiple comparison test was used to compare each condition with the control. Comparisons labeled with a single asterisk (*) indicate P < 0.05. Error bars indicate SEM, n=3. (D) Serum-starved fibroblasts were either mock infected or infected with UV-treated or untreated HCMV strain Towne (MOI = 2 TCID50/cell) , and either mock treated or treated for 10 min with 5% newborn calf serum (NCS). Samples were mechanically lysed and differentially centrifuged to separate membrane (M) and cytoplasmic (C) fractions, where subjected to Western blot analysis to monitor the expression of the indicated proteins alongside total (T) lysate.
    Figure Legend Snippet: UV-treated virus fails to inactivate AKT. (A) HCMV strain Towne was mock treated or treated with 125 mJ of UV light for indicated seconds (sec) and then used to infect fibroblasts (MOI = 2 TCID50/cell) for 12 h. A Western blot was performed to monitor for IE1 expression, using Ponceau S stain to indicate total protein loading. n = 1. (B–C) Fibroblasts were infected with mock-treated or UV-treated HCMV strain Towne virus (MOI = 2 TCID50/cell) in the presence of serum, and lysates were taken at the indicated times post-infection (in hours), hours post-infection (hpi). A Western blot was performed probing for the indicated proteins; additionally, Ponceau S staining is shown as a readout for total protein loading. (C) Detection signals from 24 hpi bands were quantified, normalized to signals from 1 hpi bands, and the arithmetic means from three independent biological replicates were plotted. A one-way ANOVA with Tukey’s multiple comparison test was used to compare each condition with the control. Comparisons labeled with a single asterisk (*) indicate P < 0.05. Error bars indicate SEM, n=3. (D) Serum-starved fibroblasts were either mock infected or infected with UV-treated or untreated HCMV strain Towne (MOI = 2 TCID50/cell) , and either mock treated or treated for 10 min with 5% newborn calf serum (NCS). Samples were mechanically lysed and differentially centrifuged to separate membrane (M) and cytoplasmic (C) fractions, where subjected to Western blot analysis to monitor the expression of the indicated proteins alongside total (T) lysate.

    Techniques Used: Virus, Western Blot, Expressing, Staining, Infection, Comparison, Labeling, Membrane

    HCMV causes degradation of insulin receptor substrate 1. (A) Model for AKT inactivation in HCMV-infected cells. HCMV UL38-mediated activation of mTORC1 leads to phosphorylation of insulin receptor substrate 1 (IRS1), inducing its degradation. Destabilization of IRS1 in turn prevents phosphoinositide 3-kinase (PI3K) recruitment to growth factor receptors (not shown), preventing AKT membrane recruitment and activation. Cartoon was generated using BioRender. (B–C) Fibroblasts were mock infected or infected with HCMV strain Towne (MOI = 2 TCID50/cell) in the presence of serum and lysates were taken at the indicated hours post-infection (hpi). Western blots were performed to monitor levels of the indicated proteins, (C) bands were quantified, and detection signals were normalized to total protein in 11 independent biological replicates; the arithmetic means of the combined results were graphed. The dotted line indicates phosphorylation levels detected from mock-infected controls at 1 hpi, the setting against which all infected timepoints are normalized. Error bars indicate SEM. n = 11. (D–E) Similar to the experiment in Fig. 1C, fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne multiplicity of infection (MOI = 2 TCID50/cell). At 3 hpi, cells were mock treated or treated with 50 nM rapamycin. At 24 hpi, cells were then either treated or mock treated with serum for 10 min, and lysates were harvested for Western blot analysis. Ponceau S staining was used to monitor total protein loading. (E) Bands were quantified, and phospho-specific antibody signals were normalized to detection signal for cognate total protein and then further normalized to “no drug” control conditions. The arithmetic mean was calculated and graphed. A one-way ANOVA with Tukey’s multiple comparison test was used to assess statistical significance, wherein *P < 0.05 and **P < 0.01. Error bars indicate SEM. n = 3 independent biological replicates.
    Figure Legend Snippet: HCMV causes degradation of insulin receptor substrate 1. (A) Model for AKT inactivation in HCMV-infected cells. HCMV UL38-mediated activation of mTORC1 leads to phosphorylation of insulin receptor substrate 1 (IRS1), inducing its degradation. Destabilization of IRS1 in turn prevents phosphoinositide 3-kinase (PI3K) recruitment to growth factor receptors (not shown), preventing AKT membrane recruitment and activation. Cartoon was generated using BioRender. (B–C) Fibroblasts were mock infected or infected with HCMV strain Towne (MOI = 2 TCID50/cell) in the presence of serum and lysates were taken at the indicated hours post-infection (hpi). Western blots were performed to monitor levels of the indicated proteins, (C) bands were quantified, and detection signals were normalized to total protein in 11 independent biological replicates; the arithmetic means of the combined results were graphed. The dotted line indicates phosphorylation levels detected from mock-infected controls at 1 hpi, the setting against which all infected timepoints are normalized. Error bars indicate SEM. n = 11. (D–E) Similar to the experiment in Fig. 1C, fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne multiplicity of infection (MOI = 2 TCID50/cell). At 3 hpi, cells were mock treated or treated with 50 nM rapamycin. At 24 hpi, cells were then either treated or mock treated with serum for 10 min, and lysates were harvested for Western blot analysis. Ponceau S staining was used to monitor total protein loading. (E) Bands were quantified, and phospho-specific antibody signals were normalized to detection signal for cognate total protein and then further normalized to “no drug” control conditions. The arithmetic mean was calculated and graphed. A one-way ANOVA with Tukey’s multiple comparison test was used to assess statistical significance, wherein *P < 0.05 and **P < 0.01. Error bars indicate SEM. n = 3 independent biological replicates.

    Techniques Used: Infection, Activation Assay, Membrane, Generated, Western Blot, Staining, Comparison

    hcmv strains towne  (ATCC)


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  • 86

    Structured Review

    ATCC hcmv strains towne
    Human cytomegalovirus <t>(HCMV)</t> prevents AKT activation in response to serum. (A) Stimulation of various receptor tyrosine kinases (RTKs) and other growth factor receptors causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, activating its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family transcription factors, and the tuberous sclerosis complex 2 (TSC2) protei. Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. (Schematic generated in BioRender.) (B) Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, and transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared with AKT (pan) antibody. (C–G) Fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain <t>Towne</t> at multiplicity of infection (MOI) of 2 (MOI = 2 TCID50/cell) for the indicated times, hours post-infection (hpi). Cells were then either treated (or mock treated) with serum for 10 min prior to lysis for Western blot analysis. (E–G) Fluorescent signals from dye-labeled secondary antibodies were quantified, and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473, and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used to visualize protein loading and confirm efficient transfer.
    Hcmv Strains Towne, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hcmv strains towne/product/ATCC
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hcmv strains towne - by Bioz Stars, 2023-11
    86/100 stars

    Images

    1) Product Images from "Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins"

    Article Title: Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins

    Journal: Journal of Virology

    doi: 10.1128/jvi.00563-23

    Human cytomegalovirus (HCMV) prevents AKT activation in response to serum. (A) Stimulation of various receptor tyrosine kinases (RTKs) and other growth factor receptors causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, activating its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family transcription factors, and the tuberous sclerosis complex 2 (TSC2) protei. Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. (Schematic generated in BioRender.) (B) Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, and transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared with AKT (pan) antibody. (C–G) Fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne at multiplicity of infection (MOI) of 2 (MOI = 2 TCID50/cell) for the indicated times, hours post-infection (hpi). Cells were then either treated (or mock treated) with serum for 10 min prior to lysis for Western blot analysis. (E–G) Fluorescent signals from dye-labeled secondary antibodies were quantified, and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473, and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used to visualize protein loading and confirm efficient transfer.
    Figure Legend Snippet: Human cytomegalovirus (HCMV) prevents AKT activation in response to serum. (A) Stimulation of various receptor tyrosine kinases (RTKs) and other growth factor receptors causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, activating its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family transcription factors, and the tuberous sclerosis complex 2 (TSC2) protei. Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. (Schematic generated in BioRender.) (B) Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, and transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared with AKT (pan) antibody. (C–G) Fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne at multiplicity of infection (MOI) of 2 (MOI = 2 TCID50/cell) for the indicated times, hours post-infection (hpi). Cells were then either treated (or mock treated) with serum for 10 min prior to lysis for Western blot analysis. (E–G) Fluorescent signals from dye-labeled secondary antibodies were quantified, and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473, and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used to visualize protein loading and confirm efficient transfer.

    Techniques Used: Activation Assay, Scaffolding, Activity Assay, Generated, Lysis, SDS Page, Western Blot, Infection, Labeling, Staining

    Membrane recruitment of AKT is defective in HCMV-infected cells. (A–B) Serum-starved fibroblasts were either mock infected or infected with HCMV strain Towne (MOI= 2 TCID50/cell) for 12 h and either mock treated or treated with 5% newborn calf serum (NCS) for 10 min. Samples were mechanically lysed and differentially centrifuged to separate the membrane (M) fraction from the cytoplasmic (C) fraction and subjugated to Western blot analysis of indicated proteins alongside total (T) lysate. Ponceau staining was used as a loading control. (B) Detection signals from AKT (pan) bands were quantified for each condition, and bands from membrane fractions were normalized to signal cognate “total lysate” bands. Results were then normalized to the “No Serum” condition, the arithmetic mean was calculated, and values were graphed. A one-way ANOVA was run using Tukey’s multiple comparison post-test for statistical analysis (*P < 0.05, ****P < 0.0001). Error bars indicate standard error of the mean (SEM). n = 4 independent biological replicates.
    Figure Legend Snippet: Membrane recruitment of AKT is defective in HCMV-infected cells. (A–B) Serum-starved fibroblasts were either mock infected or infected with HCMV strain Towne (MOI= 2 TCID50/cell) for 12 h and either mock treated or treated with 5% newborn calf serum (NCS) for 10 min. Samples were mechanically lysed and differentially centrifuged to separate the membrane (M) fraction from the cytoplasmic (C) fraction and subjugated to Western blot analysis of indicated proteins alongside total (T) lysate. Ponceau staining was used as a loading control. (B) Detection signals from AKT (pan) bands were quantified for each condition, and bands from membrane fractions were normalized to signal cognate “total lysate” bands. Results were then normalized to the “No Serum” condition, the arithmetic mean was calculated, and values were graphed. A one-way ANOVA was run using Tukey’s multiple comparison post-test for statistical analysis (*P < 0.05, ****P < 0.0001). Error bars indicate standard error of the mean (SEM). n = 4 independent biological replicates.

    Techniques Used: Membrane, Infection, Western Blot, Staining, Comparison

    UV-treated virus fails to inactivate AKT. (A) HCMV strain Towne was mock treated or treated with 125 mJ of UV light for indicated seconds (sec) and then used to infect fibroblasts (MOI = 2 TCID50/cell) for 12 h. A Western blot was performed to monitor for IE1 expression, using Ponceau S stain to indicate total protein loading. n = 1. (B–C) Fibroblasts were infected with mock-treated or UV-treated HCMV strain Towne virus (MOI = 2 TCID50/cell) in the presence of serum, and lysates were taken at the indicated times post-infection (in hours), hours post-infection (hpi). A Western blot was performed probing for the indicated proteins; additionally, Ponceau S staining is shown as a readout for total protein loading. (C) Detection signals from 24 hpi bands were quantified, normalized to signals from 1 hpi bands, and the arithmetic means from three independent biological replicates were plotted. A one-way ANOVA with Tukey’s multiple comparison test was used to compare each condition with the control. Comparisons labeled with a single asterisk (*) indicate P < 0.05. Error bars indicate SEM, n=3. (D) Serum-starved fibroblasts were either mock infected or infected with UV-treated or untreated HCMV strain Towne (MOI = 2 TCID50/cell) , and either mock treated or treated for 10 min with 5% newborn calf serum (NCS). Samples were mechanically lysed and differentially centrifuged to separate membrane (M) and cytoplasmic (C) fractions, where subjected to Western blot analysis to monitor the expression of the indicated proteins alongside total (T) lysate.
    Figure Legend Snippet: UV-treated virus fails to inactivate AKT. (A) HCMV strain Towne was mock treated or treated with 125 mJ of UV light for indicated seconds (sec) and then used to infect fibroblasts (MOI = 2 TCID50/cell) for 12 h. A Western blot was performed to monitor for IE1 expression, using Ponceau S stain to indicate total protein loading. n = 1. (B–C) Fibroblasts were infected with mock-treated or UV-treated HCMV strain Towne virus (MOI = 2 TCID50/cell) in the presence of serum, and lysates were taken at the indicated times post-infection (in hours), hours post-infection (hpi). A Western blot was performed probing for the indicated proteins; additionally, Ponceau S staining is shown as a readout for total protein loading. (C) Detection signals from 24 hpi bands were quantified, normalized to signals from 1 hpi bands, and the arithmetic means from three independent biological replicates were plotted. A one-way ANOVA with Tukey’s multiple comparison test was used to compare each condition with the control. Comparisons labeled with a single asterisk (*) indicate P < 0.05. Error bars indicate SEM, n=3. (D) Serum-starved fibroblasts were either mock infected or infected with UV-treated or untreated HCMV strain Towne (MOI = 2 TCID50/cell) , and either mock treated or treated for 10 min with 5% newborn calf serum (NCS). Samples were mechanically lysed and differentially centrifuged to separate membrane (M) and cytoplasmic (C) fractions, where subjected to Western blot analysis to monitor the expression of the indicated proteins alongside total (T) lysate.

    Techniques Used: Virus, Western Blot, Expressing, Staining, Infection, Comparison, Labeling, Membrane

    HCMV causes degradation of insulin receptor substrate 1. (A) Model for AKT inactivation in HCMV-infected cells. HCMV UL38-mediated activation of mTORC1 leads to phosphorylation of insulin receptor substrate 1 (IRS1), inducing its degradation. Destabilization of IRS1 in turn prevents phosphoinositide 3-kinase (PI3K) recruitment to growth factor receptors (not shown), preventing AKT membrane recruitment and activation. Cartoon was generated using BioRender. (B–C) Fibroblasts were mock infected or infected with HCMV strain Towne (MOI = 2 TCID50/cell) in the presence of serum and lysates were taken at the indicated hours post-infection (hpi). Western blots were performed to monitor levels of the indicated proteins, (C) bands were quantified, and detection signals were normalized to total protein in 11 independent biological replicates; the arithmetic means of the combined results were graphed. The dotted line indicates phosphorylation levels detected from mock-infected controls at 1 hpi, the setting against which all infected timepoints are normalized. Error bars indicate SEM. n = 11. (D–E) Similar to the experiment in Fig. 1C, fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne multiplicity of infection (MOI = 2 TCID50/cell). At 3 hpi, cells were mock treated or treated with 50 nM rapamycin. At 24 hpi, cells were then either treated or mock treated with serum for 10 min, and lysates were harvested for Western blot analysis. Ponceau S staining was used to monitor total protein loading. (E) Bands were quantified, and phospho-specific antibody signals were normalized to detection signal for cognate total protein and then further normalized to “no drug” control conditions. The arithmetic mean was calculated and graphed. A one-way ANOVA with Tukey’s multiple comparison test was used to assess statistical significance, wherein *P < 0.05 and **P < 0.01. Error bars indicate SEM. n = 3 independent biological replicates.
    Figure Legend Snippet: HCMV causes degradation of insulin receptor substrate 1. (A) Model for AKT inactivation in HCMV-infected cells. HCMV UL38-mediated activation of mTORC1 leads to phosphorylation of insulin receptor substrate 1 (IRS1), inducing its degradation. Destabilization of IRS1 in turn prevents phosphoinositide 3-kinase (PI3K) recruitment to growth factor receptors (not shown), preventing AKT membrane recruitment and activation. Cartoon was generated using BioRender. (B–C) Fibroblasts were mock infected or infected with HCMV strain Towne (MOI = 2 TCID50/cell) in the presence of serum and lysates were taken at the indicated hours post-infection (hpi). Western blots were performed to monitor levels of the indicated proteins, (C) bands were quantified, and detection signals were normalized to total protein in 11 independent biological replicates; the arithmetic means of the combined results were graphed. The dotted line indicates phosphorylation levels detected from mock-infected controls at 1 hpi, the setting against which all infected timepoints are normalized. Error bars indicate SEM. n = 11. (D–E) Similar to the experiment in Fig. 1C, fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne multiplicity of infection (MOI = 2 TCID50/cell). At 3 hpi, cells were mock treated or treated with 50 nM rapamycin. At 24 hpi, cells were then either treated or mock treated with serum for 10 min, and lysates were harvested for Western blot analysis. Ponceau S staining was used to monitor total protein loading. (E) Bands were quantified, and phospho-specific antibody signals were normalized to detection signal for cognate total protein and then further normalized to “no drug” control conditions. The arithmetic mean was calculated and graphed. A one-way ANOVA with Tukey’s multiple comparison test was used to assess statistical significance, wherein *P < 0.05 and **P < 0.01. Error bars indicate SEM. n = 3 independent biological replicates.

    Techniques Used: Infection, Activation Assay, Membrane, Generated, Western Blot, Staining, Comparison

    reconstructed hcmv towne strain  (ATCC)


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    ATCC reconstructed hcmv towne strain
    Reconstructed Hcmv Towne Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hcmv strains towne  (ATCC)


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    ATCC hcmv strains towne
    ( A ): Stimulation of growth factor receptor tyrosine kinases (RTK) causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, causing full activation its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family of transcription factors, and the tuberous sclerosis complex 2 protein (TSC2). Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. [Schematic generated in BioRender.] ( B ): Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared to AKT (pan) antibody. ( C-G ): Fibroblasts were serum starved overnight and either mock infected or infected with <t>HCMV</t> strain <t>Towne</t> at multiplicity of infection (MOI=2 TCID50/cell) for the indicated times, h post-infection (hpi). Cells were then either treated (or mock-treated) with serum for 10 min prior to lysis for Western blot analysis. ( E-G ): Fluorescent signals from dye labeled secondary antibodies were quantified and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473 and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40, respectively. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used visualize protein loading and confirm efficient transfer.
    Hcmv Strains Towne, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins"

    Article Title: Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins

    Journal: bioRxiv

    doi: 10.1101/2023.04.17.537203

    ( A ): Stimulation of growth factor receptor tyrosine kinases (RTK) causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, causing full activation its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family of transcription factors, and the tuberous sclerosis complex 2 protein (TSC2). Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. [Schematic generated in BioRender.] ( B ): Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared to AKT (pan) antibody. ( C-G ): Fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne at multiplicity of infection (MOI=2 TCID50/cell) for the indicated times, h post-infection (hpi). Cells were then either treated (or mock-treated) with serum for 10 min prior to lysis for Western blot analysis. ( E-G ): Fluorescent signals from dye labeled secondary antibodies were quantified and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473 and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40, respectively. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used visualize protein loading and confirm efficient transfer.
    Figure Legend Snippet: ( A ): Stimulation of growth factor receptor tyrosine kinases (RTK) causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, causing full activation its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family of transcription factors, and the tuberous sclerosis complex 2 protein (TSC2). Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. [Schematic generated in BioRender.] ( B ): Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared to AKT (pan) antibody. ( C-G ): Fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne at multiplicity of infection (MOI=2 TCID50/cell) for the indicated times, h post-infection (hpi). Cells were then either treated (or mock-treated) with serum for 10 min prior to lysis for Western blot analysis. ( E-G ): Fluorescent signals from dye labeled secondary antibodies were quantified and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473 and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40, respectively. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used visualize protein loading and confirm efficient transfer.

    Techniques Used: Scaffolding, Activation Assay, Activity Assay, Generated, Lysis, SDS Page, Western Blot, Infection, Labeling, Staining

    ( A-B ) Serum-starved fibroblasts were either mock-infected or infected with HCMV strain Towne (MOI=2 TCID50/cell) for 12 hours and either mock-treated or treated with 5% NCS (serum) for 10 minutes. Samples were mechanically lysed and differentially centrifuged to separate the membrane (M) fraction from the cytoplasmic (C) fraction and subjugated to Western blot analysis of indicated proteins alongside total (T) lysate. Ponceau staining was used as a loading control. ( B ) The AKT (pan) bands were quantified and the membrane band was normalized to the total lysate band. Quantification was then normalized to-Serum, the arithmetic mean was calculated and values were graphed. Error bars=SEM. n=4.
    Figure Legend Snippet: ( A-B ) Serum-starved fibroblasts were either mock-infected or infected with HCMV strain Towne (MOI=2 TCID50/cell) for 12 hours and either mock-treated or treated with 5% NCS (serum) for 10 minutes. Samples were mechanically lysed and differentially centrifuged to separate the membrane (M) fraction from the cytoplasmic (C) fraction and subjugated to Western blot analysis of indicated proteins alongside total (T) lysate. Ponceau staining was used as a loading control. ( B ) The AKT (pan) bands were quantified and the membrane band was normalized to the total lysate band. Quantification was then normalized to-Serum, the arithmetic mean was calculated and values were graphed. Error bars=SEM. n=4.

    Techniques Used: Infection, Western Blot, Staining

    ( A ) HCMV strain Towne was mock-treated or treated with 125mJ of UV light for indicated seconds (sec) and allowed to infect fibroblasts (MOI=2 TCID50/cell) for 12 hours. A Western blot was performed staining for IE1 with Ponceau stain used as a readout for loading. n=1. ( B-C ) Fibroblasts were infected with mock-treated or UV-treated HCMV strain Towne (MOI=2 TCID50/cell) in the presence of serum and lysates were taken at the indicated hours post-infection (hpi). A Western blot was performed probing for the indicated proteins using Ponceau stain as a readout for loading. ( C ) Bands were quantified at 24 hpi, normalized to signal at 1 hpi, and the arithmetic mean was found and graphed. Sidak statistical test were used to compare each condition to the control and labeled with asterisks (ns=P>0.05, *=P<0.05, **=P<0.01, ***=P<0.001, ****P<0.0001). Error bars=SEM. n=3. ( D ) Serum-starved fibroblasts were either mock-infected or infected with UV-treated or untreated HCMV strain Towne (MOI=2 TCID50/cell) for 12 hours and either mock-treated or treated with 5% NCS (serum) for 10 minutes. Samples were mechanically lysed and differentially centrifuged to separate the membrane (M) fraction from the cytoplasmic (C) fraction and subjugated to Western blot analysis of indicated proteins alongside total (T) lysate.
    Figure Legend Snippet: ( A ) HCMV strain Towne was mock-treated or treated with 125mJ of UV light for indicated seconds (sec) and allowed to infect fibroblasts (MOI=2 TCID50/cell) for 12 hours. A Western blot was performed staining for IE1 with Ponceau stain used as a readout for loading. n=1. ( B-C ) Fibroblasts were infected with mock-treated or UV-treated HCMV strain Towne (MOI=2 TCID50/cell) in the presence of serum and lysates were taken at the indicated hours post-infection (hpi). A Western blot was performed probing for the indicated proteins using Ponceau stain as a readout for loading. ( C ) Bands were quantified at 24 hpi, normalized to signal at 1 hpi, and the arithmetic mean was found and graphed. Sidak statistical test were used to compare each condition to the control and labeled with asterisks (ns=P>0.05, *=P<0.05, **=P<0.01, ***=P<0.001, ****P<0.0001). Error bars=SEM. n=3. ( D ) Serum-starved fibroblasts were either mock-infected or infected with UV-treated or untreated HCMV strain Towne (MOI=2 TCID50/cell) for 12 hours and either mock-treated or treated with 5% NCS (serum) for 10 minutes. Samples were mechanically lysed and differentially centrifuged to separate the membrane (M) fraction from the cytoplasmic (C) fraction and subjugated to Western blot analysis of indicated proteins alongside total (T) lysate.

    Techniques Used: Western Blot, Staining, Infection, Labeling

    ( A ) PDK1 and mTORC2 phosphorylate AKT at Thr308 and Ser473 respectively following AKT membrane recruitment. Fibroblasts were transduced with a lentivirus harboring a “tet-on” promoter controlling expression of an AKT1 gene whose PH-domain has been replaced with a myristoylation (myr) signal and genetically fused to an hemagglutinin (HA) tag. Following expression, myrAKT is embedded in the membrane simulating constitutive “membrane recruitment” and is phosphorylated as such by PDK1 and mTORC2. This experiment addresses if the activators downstream of AKT, PDK1 and mTORC2, are active during HCMV infection. Figure was created using BioRender. ( B ) Induction of myrAKT occured after overnight treatment with doxycycline followed by infection or mock infection with HCMV strain TOWNE (MOI=2 TCID50/cell) for indicated hours post infection (hpi). A Western blot was performed probing for the indicated proteins using Ponceau stain as a readout for loading. In pAKT and Total AKT blots, both endogenously expressed (Endo) AKT and myrAKT (Myr) migrated to different molecular weights due to deletion of PH-domain in myrAKT gene. ( C ) pAKT (Thr308) signal and ( D ) pAKT (Ser473) signal were quantified, normalized to AKT (pan) and subsequently normalized to 0 hpi signal at respective molecular weight. The arithmetic mean was calculated and graphed. Error bars=SEM. n=3.
    Figure Legend Snippet: ( A ) PDK1 and mTORC2 phosphorylate AKT at Thr308 and Ser473 respectively following AKT membrane recruitment. Fibroblasts were transduced with a lentivirus harboring a “tet-on” promoter controlling expression of an AKT1 gene whose PH-domain has been replaced with a myristoylation (myr) signal and genetically fused to an hemagglutinin (HA) tag. Following expression, myrAKT is embedded in the membrane simulating constitutive “membrane recruitment” and is phosphorylated as such by PDK1 and mTORC2. This experiment addresses if the activators downstream of AKT, PDK1 and mTORC2, are active during HCMV infection. Figure was created using BioRender. ( B ) Induction of myrAKT occured after overnight treatment with doxycycline followed by infection or mock infection with HCMV strain TOWNE (MOI=2 TCID50/cell) for indicated hours post infection (hpi). A Western blot was performed probing for the indicated proteins using Ponceau stain as a readout for loading. In pAKT and Total AKT blots, both endogenously expressed (Endo) AKT and myrAKT (Myr) migrated to different molecular weights due to deletion of PH-domain in myrAKT gene. ( C ) pAKT (Thr308) signal and ( D ) pAKT (Ser473) signal were quantified, normalized to AKT (pan) and subsequently normalized to 0 hpi signal at respective molecular weight. The arithmetic mean was calculated and graphed. Error bars=SEM. n=3.

    Techniques Used: Transduction, Expressing, Infection, Western Blot, Staining, Molecular Weight

    ( A ) Model for AKT inactivation in HCMV infected cells. HCMV UL38 mediated activation of mTORC1 leads to mTORC1 phosphorylation of IRS1, concomitantly inducing IRS1 degradation. This destabilization of IRS1 prevents PI3K recruitment to growth factor receptors (not shown), preventing AKT membrane recruitment and activation. Cartoon was generated using BioRender. ( B-C ) Fibroblasts were mock-infected or infected with HCMV strain Towne (MOI=2 TCID50/cell) in the presence of serum and lysates were taken at the indicated hours post-infection (hpi). A Western blot analysis detecting the indicated proteins was performed and (C) bands were quantified and phospho-specific quantification was normalized to total protein and arithmetic mean was found and graphed. The dotted line is the phosphorylation of mock-infected controls at 1 hpi, to which all infected time points are normalized to. Error bars=SEM. n=11. ( D-E ) Similar to , fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne multiplicity of infection (MOI=2 TCID50/cell). At 3 hpi, cells were mock-treated or treated with Rapamycin. At 24 hpi, cells were then either treated or mock-treated with serum for 10 minutes, and lysates were taken for Western blot analysis detection of indicated proteins. Ponceau staining was used for loading control. ( E ) Bands of the HCMV-infected cells were quantified, and phospho-specific quantification was normalized to total protein then normalized to ‘No Rapamycin’ control and arithmetic mean was found and graphed. Sidak statistical test were used to compare each condition to the control and labeled with asterisks (ns=P>0.05, *=P<0.05, **=P<0.01, ***=P<0.001, ****P<0.0001). Error bars=SEM. n=3.
    Figure Legend Snippet: ( A ) Model for AKT inactivation in HCMV infected cells. HCMV UL38 mediated activation of mTORC1 leads to mTORC1 phosphorylation of IRS1, concomitantly inducing IRS1 degradation. This destabilization of IRS1 prevents PI3K recruitment to growth factor receptors (not shown), preventing AKT membrane recruitment and activation. Cartoon was generated using BioRender. ( B-C ) Fibroblasts were mock-infected or infected with HCMV strain Towne (MOI=2 TCID50/cell) in the presence of serum and lysates were taken at the indicated hours post-infection (hpi). A Western blot analysis detecting the indicated proteins was performed and (C) bands were quantified and phospho-specific quantification was normalized to total protein and arithmetic mean was found and graphed. The dotted line is the phosphorylation of mock-infected controls at 1 hpi, to which all infected time points are normalized to. Error bars=SEM. n=11. ( D-E ) Similar to , fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne multiplicity of infection (MOI=2 TCID50/cell). At 3 hpi, cells were mock-treated or treated with Rapamycin. At 24 hpi, cells were then either treated or mock-treated with serum for 10 minutes, and lysates were taken for Western blot analysis detection of indicated proteins. Ponceau staining was used for loading control. ( E ) Bands of the HCMV-infected cells were quantified, and phospho-specific quantification was normalized to total protein then normalized to ‘No Rapamycin’ control and arithmetic mean was found and graphed. Sidak statistical test were used to compare each condition to the control and labeled with asterisks (ns=P>0.05, *=P<0.05, **=P<0.01, ***=P<0.001, ****P<0.0001). Error bars=SEM. n=3.

    Techniques Used: Infection, Activation Assay, Generated, Western Blot, Staining, Labeling

    klebsiella pneumonia strain peterborough town cricket club 10031 t  (ATCC)


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    ATCC klebsiella pneumonia strain peterborough town cricket club 10031 t
    The antimicrobial effect of concentrated bacterial cell‐free culture medium on the growth of (a) Klebsiella pneumonia strain Peterborough Town Cricket <t>Club</t> <t>10031</t> <t>T</t> and (b) Streptococcus aureus strain American Type Culture Collection 35668 T .
    Klebsiella Pneumonia Strain Peterborough Town Cricket Club 10031 T, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Synthesis of Zeolitic imidazolate frameworks‐8@ layered double hydroxide polyhedral nanocomposite with designed porous voids as an effective carrier for anti‐cancer drug‐controlled delivery"

    Article Title: Synthesis of Zeolitic imidazolate frameworks‐8@ layered double hydroxide polyhedral nanocomposite with designed porous voids as an effective carrier for anti‐cancer drug‐controlled delivery

    Journal: IET Nanobiotechnology

    doi: 10.1049/nbt2.12125

    The antimicrobial effect of concentrated bacterial cell‐free culture medium on the growth of (a) Klebsiella pneumonia strain Peterborough Town Cricket Club 10031 T and (b) Streptococcus aureus strain American Type Culture Collection 35668 T .
    Figure Legend Snippet: The antimicrobial effect of concentrated bacterial cell‐free culture medium on the growth of (a) Klebsiella pneumonia strain Peterborough Town Cricket Club 10031 T and (b) Streptococcus aureus strain American Type Culture Collection 35668 T .

    Techniques Used:

    hcmv towne strain  (ATCC)


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    ATCC hcmv towne strain
    Hcmv Towne Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hcmv towne strain  (ATCC)


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    ATCC hcmv towne strain
    Hcmv Towne Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    strain towne  (ATCC)


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    ATCC strain towne
    Strain Towne, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hcmv towne strain  (ATCC)


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    ATCC hcmv towne strain
    Hematoxylin/eosin (A-B and G) and fluorescent staining (C-F, H) of EpiGingival tissues. The tissues were either mock infected (A, C) or infected with 2 × 10 4 PFU of <t>HCMV</t> mutant ΔUS18 (G and H) and the parental <t>Towne</t> BAC (B, D, E, and F), harvested at 7 days post infection, fixed with Streck Tissue Fixative, frozen in 2-methylbutane submerged in liquid nitrogen, and cross-sectioned at 9 μm using a LEICA cryostat LC1900 sectioner, stained with either hematoxylin/eosin or DAPI, and visualized (magnification, ×400). The cells that were infected with Towne BAC and ΔUS18, which carried a GFP expression cassette, were visualized by detecting the expression of GFP (C, E, F, and H). The images of the DAPI-staining tissues (DAPI) (D) and the infected cells that expressed the GFP (GFP) (E) were used to generate the composite image (GFP+DAPI) (F). Similar composite images (GFP+DAPI) are shown in (C) and (H).
    Hcmv Towne Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Infection of human cytomegalovirus in cultured human gingival tissue"

    Article Title: Infection of human cytomegalovirus in cultured human gingival tissue

    Journal: Virology Journal

    doi: 10.1186/1743-422X-3-84

    Hematoxylin/eosin (A-B and G) and fluorescent staining (C-F, H) of EpiGingival tissues. The tissues were either mock infected (A, C) or infected with 2 × 10 4 PFU of HCMV mutant ΔUS18 (G and H) and the parental Towne BAC (B, D, E, and F), harvested at 7 days post infection, fixed with Streck Tissue Fixative, frozen in 2-methylbutane submerged in liquid nitrogen, and cross-sectioned at 9 μm using a LEICA cryostat LC1900 sectioner, stained with either hematoxylin/eosin or DAPI, and visualized (magnification, ×400). The cells that were infected with Towne BAC and ΔUS18, which carried a GFP expression cassette, were visualized by detecting the expression of GFP (C, E, F, and H). The images of the DAPI-staining tissues (DAPI) (D) and the infected cells that expressed the GFP (GFP) (E) were used to generate the composite image (GFP+DAPI) (F). Similar composite images (GFP+DAPI) are shown in (C) and (H).
    Figure Legend Snippet: Hematoxylin/eosin (A-B and G) and fluorescent staining (C-F, H) of EpiGingival tissues. The tissues were either mock infected (A, C) or infected with 2 × 10 4 PFU of HCMV mutant ΔUS18 (G and H) and the parental Towne BAC (B, D, E, and F), harvested at 7 days post infection, fixed with Streck Tissue Fixative, frozen in 2-methylbutane submerged in liquid nitrogen, and cross-sectioned at 9 μm using a LEICA cryostat LC1900 sectioner, stained with either hematoxylin/eosin or DAPI, and visualized (magnification, ×400). The cells that were infected with Towne BAC and ΔUS18, which carried a GFP expression cassette, were visualized by detecting the expression of GFP (C, E, F, and H). The images of the DAPI-staining tissues (DAPI) (D) and the infected cells that expressed the GFP (GFP) (E) were used to generate the composite image (GFP+DAPI) (F). Similar composite images (GFP+DAPI) are shown in (C) and (H).

    Techniques Used: Staining, Infection, Mutagenesis, Expressing

    Growth of different HCMV strains (Toledo, Towne, and Towne BAC ) in cultured cells (A) and cultured gingival tissues (B). In (A), human foreskin fibroblasts (HFFs) (1 × 10 6 cells) were infected with each virus at a MOI of 0.05. At 0, 2, 4, 7, 10, and 14 days post infection, cells and culture media were harvested and sonicated. In (B), the tissues were infected with 2 × 10 4 PFU of each virus at the apical surface of the tissue. At 0, 3, 6, and 10 days post infection, the tissues were harvested, suspended in a small volume of 10% milk, and sonicated. The viral titers were determined by plaque assays on HFFs. The limit of detection was 10 PFU/ml of the tissue homogenate. The values of the viral titer represent the average obtained from triplicate experiments. The standard deviation is indicated by the error bars.
    Figure Legend Snippet: Growth of different HCMV strains (Toledo, Towne, and Towne BAC ) in cultured cells (A) and cultured gingival tissues (B). In (A), human foreskin fibroblasts (HFFs) (1 × 10 6 cells) were infected with each virus at a MOI of 0.05. At 0, 2, 4, 7, 10, and 14 days post infection, cells and culture media were harvested and sonicated. In (B), the tissues were infected with 2 × 10 4 PFU of each virus at the apical surface of the tissue. At 0, 3, 6, and 10 days post infection, the tissues were harvested, suspended in a small volume of 10% milk, and sonicated. The viral titers were determined by plaque assays on HFFs. The limit of detection was 10 PFU/ml of the tissue homogenate. The values of the viral titer represent the average obtained from triplicate experiments. The standard deviation is indicated by the error bars.

    Techniques Used: Cell Culture, Infection, Sonication, Standard Deviation

    Growth of HCMV mutants ΔUS18, ΔRL9, and the parental Towne BAC in cultured cells (A) and gingival tissues (B). In (A), human foreskin fibroblasts (HFFs) (1 × 10 6 cells) were infected with each virus at a MOI of 0.05. At 0, 2, 4, 7, 10, and 14 days post infection, cells and culture media were harvested and sonicated. In (B), the tissues were infected with 2 × 10 4 PFU of each virus at the apical surface of the tissue. At 0, 3, 6, and 10 days post infection, the tissues were harvested, suspended in 10% milk, and sonicated. The viral titers were determined by plaque assays on HFFs. The limit of detection was 10 PFU/ml of the tissue homogenate. The values of the viral titer represent the average obtained from triplicate experiments. The standard deviation is indicated by the error bars.
    Figure Legend Snippet: Growth of HCMV mutants ΔUS18, ΔRL9, and the parental Towne BAC in cultured cells (A) and gingival tissues (B). In (A), human foreskin fibroblasts (HFFs) (1 × 10 6 cells) were infected with each virus at a MOI of 0.05. At 0, 2, 4, 7, 10, and 14 days post infection, cells and culture media were harvested and sonicated. In (B), the tissues were infected with 2 × 10 4 PFU of each virus at the apical surface of the tissue. At 0, 3, 6, and 10 days post infection, the tissues were harvested, suspended in 10% milk, and sonicated. The viral titers were determined by plaque assays on HFFs. The limit of detection was 10 PFU/ml of the tissue homogenate. The values of the viral titer represent the average obtained from triplicate experiments. The standard deviation is indicated by the error bars.

    Techniques Used: Cell Culture, Infection, Sonication, Standard Deviation

    Growth of HCMV in cultured cells (A) and gingival tissues (B) that were treated with different concentrations of ganciclovir. In (A), human foreskin fibroblasts (HFFs) (1 × 10 6 cells) were infected with each virus at a MOI of 0.05. At 0, 2, 4, 7, 10, and 14 days post infection, cells and culture media were harvested and sonicated. In (B), the tissues were infected with 2 × 10 4 PFU of Towne BAC at the apical surface of the tissue. At 0, 3, 6, and 10 days post infection, the tissues were harvested, suspended in 10% milk, and sonicated. Different concentrations (10 μM or 100 μM) of GCV were added to the cultured media at 24 hours post infection. The viral titers were determined by plaque assays on HFFs. The limit of detection was 10 PFU/ml of the tissue homogenate. The values of the viral titer represent the average obtained from triplicate experiments. The standard deviation is indicated by the error bars.
    Figure Legend Snippet: Growth of HCMV in cultured cells (A) and gingival tissues (B) that were treated with different concentrations of ganciclovir. In (A), human foreskin fibroblasts (HFFs) (1 × 10 6 cells) were infected with each virus at a MOI of 0.05. At 0, 2, 4, 7, 10, and 14 days post infection, cells and culture media were harvested and sonicated. In (B), the tissues were infected with 2 × 10 4 PFU of Towne BAC at the apical surface of the tissue. At 0, 3, 6, and 10 days post infection, the tissues were harvested, suspended in 10% milk, and sonicated. Different concentrations (10 μM or 100 μM) of GCV were added to the cultured media at 24 hours post infection. The viral titers were determined by plaque assays on HFFs. The limit of detection was 10 PFU/ml of the tissue homogenate. The values of the viral titer represent the average obtained from triplicate experiments. The standard deviation is indicated by the error bars.

    Techniques Used: Cell Culture, Infection, Sonication, Standard Deviation

    hcmv towne strain  (ATCC)


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    Structured Review

    ATCC hcmv towne strain
    HELs were infected with <t>HCMV</t> strain <t>Towne</t> at an MOI of 5 and intracellular HCMV miRNAs were quantitated by stem-loop RT-PCR at the indicated times post infection. Results indicate fold-changes relative to levels measured at 3 hpi. HCMV miRNAs were assigned to group 1 (A), group 2 (B) or group 3 (C) based on kinetic patterns of expression (see text for details).
    Hcmv Towne Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Comprehensive Analysis of Human Cytomegalovirus MicroRNA Expression during Lytic and Quiescent Infection"

    Article Title: Comprehensive Analysis of Human Cytomegalovirus MicroRNA Expression during Lytic and Quiescent Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088531

    HELs were infected with HCMV strain Towne at an MOI of 5 and intracellular HCMV miRNAs were quantitated by stem-loop RT-PCR at the indicated times post infection. Results indicate fold-changes relative to levels measured at 3 hpi. HCMV miRNAs were assigned to group 1 (A), group 2 (B) or group 3 (C) based on kinetic patterns of expression (see text for details).
    Figure Legend Snippet: HELs were infected with HCMV strain Towne at an MOI of 5 and intracellular HCMV miRNAs were quantitated by stem-loop RT-PCR at the indicated times post infection. Results indicate fold-changes relative to levels measured at 3 hpi. HCMV miRNAs were assigned to group 1 (A), group 2 (B) or group 3 (C) based on kinetic patterns of expression (see text for details).

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Expressing

    Expression kinetics for four HCMV miRNAs representing the three expression pattern groups are shown side-by-side to compare expression patterns in HELs vs. THP-1s (data are from experiments described in and ). The compared representatives of group 1 (miR-112 and miR-70-3p), group 2 (miR-22A-5p), and group 3 (miR-US33-3p) are shown. Note that HELs and THP-1 cells were infected with HCMV strain Towne at MOIs of 5 and 10, respectively. *P<0.05, **P<0.01.
    Figure Legend Snippet: Expression kinetics for four HCMV miRNAs representing the three expression pattern groups are shown side-by-side to compare expression patterns in HELs vs. THP-1s (data are from experiments described in and ). The compared representatives of group 1 (miR-112 and miR-70-3p), group 2 (miR-22A-5p), and group 3 (miR-US33-3p) are shown. Note that HELs and THP-1 cells were infected with HCMV strain Towne at MOIs of 5 and 10, respectively. *P<0.05, **P<0.01.

    Techniques Used: Expressing, Infection

    The THP-1 and d-THP-1 cells were infected with Towne at an MOI of 10, and cells were harvest at the indicated times post infection, respectively. Expression kinetics for four HCMV miRNAs (miR-112 and miR-70-3p, miR-22A-5p, and miR-US33-3p) is shown side-by-side to compare expression patterns in THP-1 vs. d-THP-1(data are from experiments described in and ). The different fold change of time points were shown above, **P<0.01, *P<0.05.
    Figure Legend Snippet: The THP-1 and d-THP-1 cells were infected with Towne at an MOI of 10, and cells were harvest at the indicated times post infection, respectively. Expression kinetics for four HCMV miRNAs (miR-112 and miR-70-3p, miR-22A-5p, and miR-US33-3p) is shown side-by-side to compare expression patterns in THP-1 vs. d-THP-1(data are from experiments described in and ). The different fold change of time points were shown above, **P<0.01, *P<0.05.

    Techniques Used: Infection, Expressing

    (A) HCMV miRNA levels were quantitated by stem-loop RT-PCR in HEL cells 48 h after transduction with empty vector lentivirus (control) or with lentiviruses engineered to express miR-UL22A, miR-UL70, miR-US33, or 48 hpi of HELs with HCMV strain Towne (MOI = 5). Results represent fold-differences for each miRNA relative to control. (B) Levels of IE1, IE2, UL44, or gB were determined by western blotting. Actin serves as a loading control. Control lentivirus (−) or lentiviruses expressing according miRNAs (+) are indicated. (C) HELs were infected with HCMV strain Towne at an MOI of 0.01 48 h after transduction with control lentivirus or lentivirus expressing miR-US33. At the times indicated post infection HCMV genome copy numbers were determined by qPCR. (D) HELs were infected with HCMV strain Towne at an MOI of 0.01 48 h after transduction with control lentivirus or lentivirus expressing miR-US33 Infectious virus titers in supernatants at 96 and 144 hpi were determined. Inhibition efficiencies are % changes in virus titers in medium from HCMV-infected miR-US33-transduced cells vs. control-transduced cells. **P<0.01, *P<0.05.
    Figure Legend Snippet: (A) HCMV miRNA levels were quantitated by stem-loop RT-PCR in HEL cells 48 h after transduction with empty vector lentivirus (control) or with lentiviruses engineered to express miR-UL22A, miR-UL70, miR-US33, or 48 hpi of HELs with HCMV strain Towne (MOI = 5). Results represent fold-differences for each miRNA relative to control. (B) Levels of IE1, IE2, UL44, or gB were determined by western blotting. Actin serves as a loading control. Control lentivirus (−) or lentiviruses expressing according miRNAs (+) are indicated. (C) HELs were infected with HCMV strain Towne at an MOI of 0.01 48 h after transduction with control lentivirus or lentivirus expressing miR-US33. At the times indicated post infection HCMV genome copy numbers were determined by qPCR. (D) HELs were infected with HCMV strain Towne at an MOI of 0.01 48 h after transduction with control lentivirus or lentivirus expressing miR-US33 Infectious virus titers in supernatants at 96 and 144 hpi were determined. Inhibition efficiencies are % changes in virus titers in medium from HCMV-infected miR-US33-transduced cells vs. control-transduced cells. **P<0.01, *P<0.05.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transduction, Plasmid Preparation, Western Blot, Expressing, Infection, Inhibition

    (A) Complementarity of miR-US33 with its putative target sequence in US29 ; the miR-US33 “seed” sequence is underlined. (B) US29 sequences are a target of miR-US33. A plasmid expressing luciferase from a transcript containing the putative miR-US33 target sequence from US29 was co-transfected into 293 cells with a plasmid expressing miR-US33 or an empty vector control plasmid. Each transfection also included a β-gal-expressing plasmid to normalize transfection efficiencies. Luciferase and β-gal activities were measured 48 h post transfection and β-gal activities were used to normalize the luciferase activities. (C) Regulation of US29 mRNA by miR-US33 during HCMV infection. HELs were infected with HCMV strain Towne at an MOI of 0.01 48 h after transduction with control lentivirus or lentivirus expressing miR-US33 and at the indicated hpi US29 mRNA levels were quantitated by RT-PCR. **P<0.01.
    Figure Legend Snippet: (A) Complementarity of miR-US33 with its putative target sequence in US29 ; the miR-US33 “seed” sequence is underlined. (B) US29 sequences are a target of miR-US33. A plasmid expressing luciferase from a transcript containing the putative miR-US33 target sequence from US29 was co-transfected into 293 cells with a plasmid expressing miR-US33 or an empty vector control plasmid. Each transfection also included a β-gal-expressing plasmid to normalize transfection efficiencies. Luciferase and β-gal activities were measured 48 h post transfection and β-gal activities were used to normalize the luciferase activities. (C) Regulation of US29 mRNA by miR-US33 during HCMV infection. HELs were infected with HCMV strain Towne at an MOI of 0.01 48 h after transduction with control lentivirus or lentivirus expressing miR-US33 and at the indicated hpi US29 mRNA levels were quantitated by RT-PCR. **P<0.01.

    Techniques Used: Sequencing, Plasmid Preparation, Expressing, Luciferase, Transfection, Infection, Transduction, Reverse Transcription Polymerase Chain Reaction

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    ATCC strain towne
    Strain Towne, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC hcmv strains towne
    Human cytomegalovirus <t>(HCMV)</t> prevents AKT activation in response to serum. (A) Stimulation of various receptor tyrosine kinases (RTKs) and other growth factor receptors causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, activating its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family transcription factors, and the tuberous sclerosis complex 2 (TSC2) protei. Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. (Schematic generated in BioRender.) (B) Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, and transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared with AKT (pan) antibody. (C–G) Fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain <t>Towne</t> at multiplicity of infection (MOI) of 2 (MOI = 2 TCID50/cell) for the indicated times, hours post-infection (hpi). Cells were then either treated (or mock treated) with serum for 10 min prior to lysis for Western blot analysis. (E–G) Fluorescent signals from dye-labeled secondary antibodies were quantified, and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473, and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used to visualize protein loading and confirm efficient transfer.
    Hcmv Strains Towne, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC reconstructed hcmv towne strain
    Human cytomegalovirus <t>(HCMV)</t> prevents AKT activation in response to serum. (A) Stimulation of various receptor tyrosine kinases (RTKs) and other growth factor receptors causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, activating its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family transcription factors, and the tuberous sclerosis complex 2 (TSC2) protei. Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. (Schematic generated in BioRender.) (B) Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, and transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared with AKT (pan) antibody. (C–G) Fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain <t>Towne</t> at multiplicity of infection (MOI) of 2 (MOI = 2 TCID50/cell) for the indicated times, hours post-infection (hpi). Cells were then either treated (or mock treated) with serum for 10 min prior to lysis for Western blot analysis. (E–G) Fluorescent signals from dye-labeled secondary antibodies were quantified, and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473, and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used to visualize protein loading and confirm efficient transfer.
    Reconstructed Hcmv Towne Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC klebsiella pneumonia strain peterborough town cricket club 10031 t
    The antimicrobial effect of concentrated bacterial cell‐free culture medium on the growth of (a) Klebsiella pneumonia strain Peterborough Town Cricket <t>Club</t> <t>10031</t> <t>T</t> and (b) Streptococcus aureus strain American Type Culture Collection 35668 T .
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    ATCC hcmv towne strain
    The antimicrobial effect of concentrated bacterial cell‐free culture medium on the growth of (a) Klebsiella pneumonia strain Peterborough Town Cricket <t>Club</t> <t>10031</t> <t>T</t> and (b) Streptococcus aureus strain American Type Culture Collection 35668 T .
    Hcmv Towne Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Human cytomegalovirus (HCMV) prevents AKT activation in response to serum. (A) Stimulation of various receptor tyrosine kinases (RTKs) and other growth factor receptors causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, activating its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family transcription factors, and the tuberous sclerosis complex 2 (TSC2) protei. Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. (Schematic generated in BioRender.) (B) Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, and transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared with AKT (pan) antibody. (C–G) Fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne at multiplicity of infection (MOI) of 2 (MOI = 2 TCID50/cell) for the indicated times, hours post-infection (hpi). Cells were then either treated (or mock treated) with serum for 10 min prior to lysis for Western blot analysis. (E–G) Fluorescent signals from dye-labeled secondary antibodies were quantified, and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473, and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used to visualize protein loading and confirm efficient transfer.

    Journal: Journal of Virology

    Article Title: Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins

    doi: 10.1128/jvi.00563-23

    Figure Lengend Snippet: Human cytomegalovirus (HCMV) prevents AKT activation in response to serum. (A) Stimulation of various receptor tyrosine kinases (RTKs) and other growth factor receptors causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, activating its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family transcription factors, and the tuberous sclerosis complex 2 (TSC2) protei. Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. (Schematic generated in BioRender.) (B) Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, and transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared with AKT (pan) antibody. (C–G) Fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne at multiplicity of infection (MOI) of 2 (MOI = 2 TCID50/cell) for the indicated times, hours post-infection (hpi). Cells were then either treated (or mock treated) with serum for 10 min prior to lysis for Western blot analysis. (E–G) Fluorescent signals from dye-labeled secondary antibodies were quantified, and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473, and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used to visualize protein loading and confirm efficient transfer.

    Article Snippet: Stocks of HCMV strains Towne (ATCC VR-977) and parental AD169rv ( 99 ) were produced by low MOI infection of HFFTs (MOI of 0.01 TCID50/cell).

    Techniques: Activation Assay, Scaffolding, Activity Assay, Generated, Lysis, SDS Page, Western Blot, Infection, Labeling, Staining

    Membrane recruitment of AKT is defective in HCMV-infected cells. (A–B) Serum-starved fibroblasts were either mock infected or infected with HCMV strain Towne (MOI= 2 TCID50/cell) for 12 h and either mock treated or treated with 5% newborn calf serum (NCS) for 10 min. Samples were mechanically lysed and differentially centrifuged to separate the membrane (M) fraction from the cytoplasmic (C) fraction and subjugated to Western blot analysis of indicated proteins alongside total (T) lysate. Ponceau staining was used as a loading control. (B) Detection signals from AKT (pan) bands were quantified for each condition, and bands from membrane fractions were normalized to signal cognate “total lysate” bands. Results were then normalized to the “No Serum” condition, the arithmetic mean was calculated, and values were graphed. A one-way ANOVA was run using Tukey’s multiple comparison post-test for statistical analysis (*P < 0.05, ****P < 0.0001). Error bars indicate standard error of the mean (SEM). n = 4 independent biological replicates.

    Journal: Journal of Virology

    Article Title: Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins

    doi: 10.1128/jvi.00563-23

    Figure Lengend Snippet: Membrane recruitment of AKT is defective in HCMV-infected cells. (A–B) Serum-starved fibroblasts were either mock infected or infected with HCMV strain Towne (MOI= 2 TCID50/cell) for 12 h and either mock treated or treated with 5% newborn calf serum (NCS) for 10 min. Samples were mechanically lysed and differentially centrifuged to separate the membrane (M) fraction from the cytoplasmic (C) fraction and subjugated to Western blot analysis of indicated proteins alongside total (T) lysate. Ponceau staining was used as a loading control. (B) Detection signals from AKT (pan) bands were quantified for each condition, and bands from membrane fractions were normalized to signal cognate “total lysate” bands. Results were then normalized to the “No Serum” condition, the arithmetic mean was calculated, and values were graphed. A one-way ANOVA was run using Tukey’s multiple comparison post-test for statistical analysis (*P < 0.05, ****P < 0.0001). Error bars indicate standard error of the mean (SEM). n = 4 independent biological replicates.

    Article Snippet: Stocks of HCMV strains Towne (ATCC VR-977) and parental AD169rv ( 99 ) were produced by low MOI infection of HFFTs (MOI of 0.01 TCID50/cell).

    Techniques: Membrane, Infection, Western Blot, Staining, Comparison

    UV-treated virus fails to inactivate AKT. (A) HCMV strain Towne was mock treated or treated with 125 mJ of UV light for indicated seconds (sec) and then used to infect fibroblasts (MOI = 2 TCID50/cell) for 12 h. A Western blot was performed to monitor for IE1 expression, using Ponceau S stain to indicate total protein loading. n = 1. (B–C) Fibroblasts were infected with mock-treated or UV-treated HCMV strain Towne virus (MOI = 2 TCID50/cell) in the presence of serum, and lysates were taken at the indicated times post-infection (in hours), hours post-infection (hpi). A Western blot was performed probing for the indicated proteins; additionally, Ponceau S staining is shown as a readout for total protein loading. (C) Detection signals from 24 hpi bands were quantified, normalized to signals from 1 hpi bands, and the arithmetic means from three independent biological replicates were plotted. A one-way ANOVA with Tukey’s multiple comparison test was used to compare each condition with the control. Comparisons labeled with a single asterisk (*) indicate P < 0.05. Error bars indicate SEM, n=3. (D) Serum-starved fibroblasts were either mock infected or infected with UV-treated or untreated HCMV strain Towne (MOI = 2 TCID50/cell) , and either mock treated or treated for 10 min with 5% newborn calf serum (NCS). Samples were mechanically lysed and differentially centrifuged to separate membrane (M) and cytoplasmic (C) fractions, where subjected to Western blot analysis to monitor the expression of the indicated proteins alongside total (T) lysate.

    Journal: Journal of Virology

    Article Title: Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins

    doi: 10.1128/jvi.00563-23

    Figure Lengend Snippet: UV-treated virus fails to inactivate AKT. (A) HCMV strain Towne was mock treated or treated with 125 mJ of UV light for indicated seconds (sec) and then used to infect fibroblasts (MOI = 2 TCID50/cell) for 12 h. A Western blot was performed to monitor for IE1 expression, using Ponceau S stain to indicate total protein loading. n = 1. (B–C) Fibroblasts were infected with mock-treated or UV-treated HCMV strain Towne virus (MOI = 2 TCID50/cell) in the presence of serum, and lysates were taken at the indicated times post-infection (in hours), hours post-infection (hpi). A Western blot was performed probing for the indicated proteins; additionally, Ponceau S staining is shown as a readout for total protein loading. (C) Detection signals from 24 hpi bands were quantified, normalized to signals from 1 hpi bands, and the arithmetic means from three independent biological replicates were plotted. A one-way ANOVA with Tukey’s multiple comparison test was used to compare each condition with the control. Comparisons labeled with a single asterisk (*) indicate P < 0.05. Error bars indicate SEM, n=3. (D) Serum-starved fibroblasts were either mock infected or infected with UV-treated or untreated HCMV strain Towne (MOI = 2 TCID50/cell) , and either mock treated or treated for 10 min with 5% newborn calf serum (NCS). Samples were mechanically lysed and differentially centrifuged to separate membrane (M) and cytoplasmic (C) fractions, where subjected to Western blot analysis to monitor the expression of the indicated proteins alongside total (T) lysate.

    Article Snippet: Stocks of HCMV strains Towne (ATCC VR-977) and parental AD169rv ( 99 ) were produced by low MOI infection of HFFTs (MOI of 0.01 TCID50/cell).

    Techniques: Virus, Western Blot, Expressing, Staining, Infection, Comparison, Labeling, Membrane

    HCMV causes degradation of insulin receptor substrate 1. (A) Model for AKT inactivation in HCMV-infected cells. HCMV UL38-mediated activation of mTORC1 leads to phosphorylation of insulin receptor substrate 1 (IRS1), inducing its degradation. Destabilization of IRS1 in turn prevents phosphoinositide 3-kinase (PI3K) recruitment to growth factor receptors (not shown), preventing AKT membrane recruitment and activation. Cartoon was generated using BioRender. (B–C) Fibroblasts were mock infected or infected with HCMV strain Towne (MOI = 2 TCID50/cell) in the presence of serum and lysates were taken at the indicated hours post-infection (hpi). Western blots were performed to monitor levels of the indicated proteins, (C) bands were quantified, and detection signals were normalized to total protein in 11 independent biological replicates; the arithmetic means of the combined results were graphed. The dotted line indicates phosphorylation levels detected from mock-infected controls at 1 hpi, the setting against which all infected timepoints are normalized. Error bars indicate SEM. n = 11. (D–E) Similar to the experiment in Fig. 1C, fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne multiplicity of infection (MOI = 2 TCID50/cell). At 3 hpi, cells were mock treated or treated with 50 nM rapamycin. At 24 hpi, cells were then either treated or mock treated with serum for 10 min, and lysates were harvested for Western blot analysis. Ponceau S staining was used to monitor total protein loading. (E) Bands were quantified, and phospho-specific antibody signals were normalized to detection signal for cognate total protein and then further normalized to “no drug” control conditions. The arithmetic mean was calculated and graphed. A one-way ANOVA with Tukey’s multiple comparison test was used to assess statistical significance, wherein *P < 0.05 and **P < 0.01. Error bars indicate SEM. n = 3 independent biological replicates.

    Journal: Journal of Virology

    Article Title: Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins

    doi: 10.1128/jvi.00563-23

    Figure Lengend Snippet: HCMV causes degradation of insulin receptor substrate 1. (A) Model for AKT inactivation in HCMV-infected cells. HCMV UL38-mediated activation of mTORC1 leads to phosphorylation of insulin receptor substrate 1 (IRS1), inducing its degradation. Destabilization of IRS1 in turn prevents phosphoinositide 3-kinase (PI3K) recruitment to growth factor receptors (not shown), preventing AKT membrane recruitment and activation. Cartoon was generated using BioRender. (B–C) Fibroblasts were mock infected or infected with HCMV strain Towne (MOI = 2 TCID50/cell) in the presence of serum and lysates were taken at the indicated hours post-infection (hpi). Western blots were performed to monitor levels of the indicated proteins, (C) bands were quantified, and detection signals were normalized to total protein in 11 independent biological replicates; the arithmetic means of the combined results were graphed. The dotted line indicates phosphorylation levels detected from mock-infected controls at 1 hpi, the setting against which all infected timepoints are normalized. Error bars indicate SEM. n = 11. (D–E) Similar to the experiment in Fig. 1C, fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne multiplicity of infection (MOI = 2 TCID50/cell). At 3 hpi, cells were mock treated or treated with 50 nM rapamycin. At 24 hpi, cells were then either treated or mock treated with serum for 10 min, and lysates were harvested for Western blot analysis. Ponceau S staining was used to monitor total protein loading. (E) Bands were quantified, and phospho-specific antibody signals were normalized to detection signal for cognate total protein and then further normalized to “no drug” control conditions. The arithmetic mean was calculated and graphed. A one-way ANOVA with Tukey’s multiple comparison test was used to assess statistical significance, wherein *P < 0.05 and **P < 0.01. Error bars indicate SEM. n = 3 independent biological replicates.

    Article Snippet: Stocks of HCMV strains Towne (ATCC VR-977) and parental AD169rv ( 99 ) were produced by low MOI infection of HFFTs (MOI of 0.01 TCID50/cell).

    Techniques: Infection, Activation Assay, Membrane, Generated, Western Blot, Staining, Comparison

    The antimicrobial effect of concentrated bacterial cell‐free culture medium on the growth of (a) Klebsiella pneumonia strain Peterborough Town Cricket Club 10031 T and (b) Streptococcus aureus strain American Type Culture Collection 35668 T .

    Journal: IET Nanobiotechnology

    Article Title: Synthesis of Zeolitic imidazolate frameworks‐8@ layered double hydroxide polyhedral nanocomposite with designed porous voids as an effective carrier for anti‐cancer drug‐controlled delivery

    doi: 10.1049/nbt2.12125

    Figure Lengend Snippet: The antimicrobial effect of concentrated bacterial cell‐free culture medium on the growth of (a) Klebsiella pneumonia strain Peterborough Town Cricket Club 10031 T and (b) Streptococcus aureus strain American Type Culture Collection 35668 T .

    Article Snippet: In the current study, an inhibition zone was observed for two pathogenic bacteria including Klebsiella pneumonia strain Peterborough Town Cricket Club 10031 T and Staphylococcus aureus strain American Type Culture Collection 35668 T (Figure ), while others were observed without any inhibition zone.

    Techniques: