hek 293 a human embryonic kidney cell line  (ATCC)


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    ATCC hek 293 a human embryonic kidney cell line
    Phosphorylation-independent MAb production and epitope mapping. (A) Reactivity of MAbs with TDP-43-Flag recombinant protein of about 46 kDa (lane 1) overexpressed in <t>HEK</t> <t>293</t> cells by Western blot. Anti-Flag MAb (Flag) was used as the positive control antibody. Leucine-rich repeat-containing protein 15 (LRRC15)-His fusion protein (lane 2) served as the negative control, which was revealed by anti-His MAb (HIS). (B) Schematic representation of the TDP-43 fragments employed for epitope mapping. (C) Mapping of TDP-43 domains with epitopes recognized by the 10 generated MAbs. This mapping was performed across the entire length of TDP-43 using immunoblot and indirect peptide ELISA. RRM, RNA-recognition motif; NLS, bipartite nuclear localization signal; NES, nuclear export signal.
    Hek 293 A Human Embryonic Kidney Cell Line, 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 "Production and characterization of novel monoclonal antibodies against pathological human TDP-43 proteins"

    Article Title: Production and characterization of novel monoclonal antibodies against pathological human TDP-43 proteins

    Journal: Journal of Neuropathology and Experimental Neurology

    doi: 10.1093/jnen/nlae042

    Phosphorylation-independent MAb production and epitope mapping. (A) Reactivity of MAbs with TDP-43-Flag recombinant protein of about 46 kDa (lane 1) overexpressed in HEK 293 cells by Western blot. Anti-Flag MAb (Flag) was used as the positive control antibody. Leucine-rich repeat-containing protein 15 (LRRC15)-His fusion protein (lane 2) served as the negative control, which was revealed by anti-His MAb (HIS). (B) Schematic representation of the TDP-43 fragments employed for epitope mapping. (C) Mapping of TDP-43 domains with epitopes recognized by the 10 generated MAbs. This mapping was performed across the entire length of TDP-43 using immunoblot and indirect peptide ELISA. RRM, RNA-recognition motif; NLS, bipartite nuclear localization signal; NES, nuclear export signal.
    Figure Legend Snippet: Phosphorylation-independent MAb production and epitope mapping. (A) Reactivity of MAbs with TDP-43-Flag recombinant protein of about 46 kDa (lane 1) overexpressed in HEK 293 cells by Western blot. Anti-Flag MAb (Flag) was used as the positive control antibody. Leucine-rich repeat-containing protein 15 (LRRC15)-His fusion protein (lane 2) served as the negative control, which was revealed by anti-His MAb (HIS). (B) Schematic representation of the TDP-43 fragments employed for epitope mapping. (C) Mapping of TDP-43 domains with epitopes recognized by the 10 generated MAbs. This mapping was performed across the entire length of TDP-43 using immunoblot and indirect peptide ELISA. RRM, RNA-recognition motif; NLS, bipartite nuclear localization signal; NES, nuclear export signal.

    Techniques Used: Recombinant, Western Blot, Positive Control, Negative Control, Generated, Peptide ELISA

    embryonic kidney cell line 293 t  (ATCC)


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    ATCC embryonic kidney cell line 293 t
    Embryonic Kidney Cell Line 293 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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    flp in t rex human embryonic kidney 293 hek293 cell line  (Thermo Fisher)


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    Thermo Fisher flp in t rex human embryonic kidney 293 hek293 cell line
    A Western blotting to confirm GTPBP8 knock-out. Whole-cell lysates from <t>HEK293,</t> GTPBP8 KO1 and GTPBP8 KO2 were used for SDS-PAGE analysis. GTPBP8 antibody was used to detect GTPBP8 endogenous levels; SDHA antibody was used as a loading control. B Growth rates of GTPBP8 knock-out cells in galactose medium. HEK293 (black), GTPBP8 KO1 (red), and GTPBP8 KO2 (yellow) were cultured in DMEM containing 0.9 g/l galactose ( n = 3 independent experiments). The mean average cell number per each time point is indicated, and error bars = ± 1 standard deviation (SD). Statistical analyses were performed using the Student’s two-tailed t -test. P -values and source data are provided in the Source Data file. C Western blotting to assess OxPhos complexes steady-state levels in GTPBP8 knock-out cell lines. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were resolved via SDS-PAGE. Membranes were probed using the OxPhos antibody cocktail against nuclear-encoded proteins of complexes V (ATP5A) and I (NDUFB8) and mtDNA-encoded complex IV protein MTCO2. GAPDH was used as a loading control. D LFQ mass spectrometry analyses of OxPhos complexes in GTPBP8 knock-out cell lines compared to HEK293 ( n = 3 independent experiments). Log 2 (FC) of GTPBP8 KO1 vs HEK293 (red) and GTPBP8 KO2 vs HEK293 (yellow) is represented on the y -axis for each OxPhos complex. Each dot refers to a protein, which constitutes the specific complex. A summary of boxplots is provided in a Source Data file. All the log 2 (FC) values are listed in Supplementary Data . E Assessment of OxPhos complex I and complex IV activities in GTPBP8 knock-out cell lines compared to control. Mitochondrial lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 cell lines were resolved by BN-PAGE and stained using complex I (CI) and complex IV (CIV) -specific substrates ( n = 1 independent experiment). Coomassie staining was used as a loading control. SC supercomplexes. Source data are provided as a Source Data file.
    Flp In T Rex Human Embryonic Kidney 293 Hek293 Cell Line, supplied by Thermo Fisher, 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 "GTPBP8 plays a role in mitoribosome formation in human mitochondria"

    Article Title: GTPBP8 plays a role in mitoribosome formation in human mitochondria

    Journal: Nature Communications

    doi: 10.1038/s41467-024-50011-x

    A Western blotting to confirm GTPBP8 knock-out. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were used for SDS-PAGE analysis. GTPBP8 antibody was used to detect GTPBP8 endogenous levels; SDHA antibody was used as a loading control. B Growth rates of GTPBP8 knock-out cells in galactose medium. HEK293 (black), GTPBP8 KO1 (red), and GTPBP8 KO2 (yellow) were cultured in DMEM containing 0.9 g/l galactose ( n = 3 independent experiments). The mean average cell number per each time point is indicated, and error bars = ± 1 standard deviation (SD). Statistical analyses were performed using the Student’s two-tailed t -test. P -values and source data are provided in the Source Data file. C Western blotting to assess OxPhos complexes steady-state levels in GTPBP8 knock-out cell lines. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were resolved via SDS-PAGE. Membranes were probed using the OxPhos antibody cocktail against nuclear-encoded proteins of complexes V (ATP5A) and I (NDUFB8) and mtDNA-encoded complex IV protein MTCO2. GAPDH was used as a loading control. D LFQ mass spectrometry analyses of OxPhos complexes in GTPBP8 knock-out cell lines compared to HEK293 ( n = 3 independent experiments). Log 2 (FC) of GTPBP8 KO1 vs HEK293 (red) and GTPBP8 KO2 vs HEK293 (yellow) is represented on the y -axis for each OxPhos complex. Each dot refers to a protein, which constitutes the specific complex. A summary of boxplots is provided in a Source Data file. All the log 2 (FC) values are listed in Supplementary Data . E Assessment of OxPhos complex I and complex IV activities in GTPBP8 knock-out cell lines compared to control. Mitochondrial lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 cell lines were resolved by BN-PAGE and stained using complex I (CI) and complex IV (CIV) -specific substrates ( n = 1 independent experiment). Coomassie staining was used as a loading control. SC supercomplexes. Source data are provided as a Source Data file.
    Figure Legend Snippet: A Western blotting to confirm GTPBP8 knock-out. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were used for SDS-PAGE analysis. GTPBP8 antibody was used to detect GTPBP8 endogenous levels; SDHA antibody was used as a loading control. B Growth rates of GTPBP8 knock-out cells in galactose medium. HEK293 (black), GTPBP8 KO1 (red), and GTPBP8 KO2 (yellow) were cultured in DMEM containing 0.9 g/l galactose ( n = 3 independent experiments). The mean average cell number per each time point is indicated, and error bars = ± 1 standard deviation (SD). Statistical analyses were performed using the Student’s two-tailed t -test. P -values and source data are provided in the Source Data file. C Western blotting to assess OxPhos complexes steady-state levels in GTPBP8 knock-out cell lines. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were resolved via SDS-PAGE. Membranes were probed using the OxPhos antibody cocktail against nuclear-encoded proteins of complexes V (ATP5A) and I (NDUFB8) and mtDNA-encoded complex IV protein MTCO2. GAPDH was used as a loading control. D LFQ mass spectrometry analyses of OxPhos complexes in GTPBP8 knock-out cell lines compared to HEK293 ( n = 3 independent experiments). Log 2 (FC) of GTPBP8 KO1 vs HEK293 (red) and GTPBP8 KO2 vs HEK293 (yellow) is represented on the y -axis for each OxPhos complex. Each dot refers to a protein, which constitutes the specific complex. A summary of boxplots is provided in a Source Data file. All the log 2 (FC) values are listed in Supplementary Data . E Assessment of OxPhos complex I and complex IV activities in GTPBP8 knock-out cell lines compared to control. Mitochondrial lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 cell lines were resolved by BN-PAGE and stained using complex I (CI) and complex IV (CIV) -specific substrates ( n = 1 independent experiment). Coomassie staining was used as a loading control. SC supercomplexes. Source data are provided as a Source Data file.

    Techniques Used: Western Blot, Knock-Out, SDS Page, Control, Cell Culture, Standard Deviation, Two Tailed Test, Mass Spectrometry, Staining

    A In vivo translation assay of GTPBP8 KO1 and GTPBP8 KO2 cell lines compared to HEK293. Cells were labeled with a [ 35 S]-methionine and cysteine mix upon cytosolic translation inhibition. Whole cell lysates were resolved by SDS-PAGE and the signal was later detected via autoradiography. Loading was determined using Coomassie staining ( n = 3 independent experiments). B Mitoribosome profiling analysis of GTPBP8 KO1 compared to HEK293. The ratio of the mitoribosome-protected fragments (RPFs) in GTPBP8 KO1 vs control for each mitochondrial transcript is represented on the y -axis. The data were determined via MitoRiboSeq and refer to a single experiment. C Quantitative real-time PCR of mt-mRNAs and mt-rRNAs steady-state levels in HEK293 cells depleted of GTPBP8 compared to control. Total RNA isolated from HEK293, GTPBP8 KO1 and GTPBP8 KO2 was used ( n = 3 biological replicates, data presented as mean values ± 1 SD). Student’s two-tailed t -test was performed. P -values and source data are provided in a Source Data file. The significance cut-off was set at p < 0.05, with * p < 0.05, ** p < 0.01, and *** p < 0.001. Source data are provided as a Source Data file.
    Figure Legend Snippet: A In vivo translation assay of GTPBP8 KO1 and GTPBP8 KO2 cell lines compared to HEK293. Cells were labeled with a [ 35 S]-methionine and cysteine mix upon cytosolic translation inhibition. Whole cell lysates were resolved by SDS-PAGE and the signal was later detected via autoradiography. Loading was determined using Coomassie staining ( n = 3 independent experiments). B Mitoribosome profiling analysis of GTPBP8 KO1 compared to HEK293. The ratio of the mitoribosome-protected fragments (RPFs) in GTPBP8 KO1 vs control for each mitochondrial transcript is represented on the y -axis. The data were determined via MitoRiboSeq and refer to a single experiment. C Quantitative real-time PCR of mt-mRNAs and mt-rRNAs steady-state levels in HEK293 cells depleted of GTPBP8 compared to control. Total RNA isolated from HEK293, GTPBP8 KO1 and GTPBP8 KO2 was used ( n = 3 biological replicates, data presented as mean values ± 1 SD). Student’s two-tailed t -test was performed. P -values and source data are provided in a Source Data file. The significance cut-off was set at p < 0.05, with * p < 0.05, ** p < 0.01, and *** p < 0.001. Source data are provided as a Source Data file.

    Techniques Used: In Vivo, Labeling, Inhibition, SDS Page, Autoradiography, Staining, Control, Real-time Polymerase Chain Reaction, Isolation, Two Tailed Test

    A Western blotting analyses of the mt-LSU and mt-SSU MRPs and selected assembly factors steady-state levels in GTPBP8-depleted cells. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were resolved via SDS-PAGE and membranes were blotted with antibodies against GTPBP8, mt-LSU MRPs (mL37, uL3m, bL28m, mL49, uL12m, uL15m), mt-SSU MRPs (uS15m, uS16m, uS17m, mS22, mS37) and others (RBFA, GTPBP10, MRM3, TRMT10C, MTG1). SDHA was used as a loading control. B Quantification of MRPs steady-state levels in GTPBP8 knock-out cells compared to control HEK293. Quantification from western blotting ( n = 3 biological replicates) was performed using the Image Lab software and protein signals were normalized to SDHA. Student’s two-tailed t -test was performed. Source data and P -values are provided in a Source Data file. C LFQ mass spectrometry analyses of MitoCarta3.0 mito-pathways in GTPBP8 knock-out cell lines compared to HEK293 ( n = 3 independent experiments). Log 2 (FC) of GTPBP8 KO1 vs HEK293 (red) and GTPBP8 KO2 vs HEK293 (yellow) is represented on the y -axis. Each dot refers to a protein belonging to mt-LSU MRPs, mt-LSU assembly factors, mt-SSU MRPs, mt-SSU assembly factors, mt-RNA processing proteins, mt-transcription and mt-translation pathways. A summary of boxplots is provided in a Supplementary Data File. The complete list of log 2 (FC) values is presented in Supplementary Data . D Sucrose gradient centrifugation analysis to assess mitoribosome sedimentation patterns in GTPBP8 KO1 and GTPBP8 KO2 compared to HEK293 control cells. Mitochondrial lysates were loaded onto 10–30 % isokinetic sucrose gradients and obtained fractions were analyzed via western blotting. Membranes were probed for mt-LSU MRP mL37 and mt-SSU MRP uS15m. Below, quantification using Image Lab software of mL37 and uS15m in the monosome fractions from independent replicates is represented ( n = 3 independent experiments). The samples derived from the same experiment were processed in parallel. The y -axis represents the signal detected in the monosome fraction normalized for the total signal of each sample’s gradient. Data are presented as mean values +/− SD. Student’s two-tailed t -test was performed. P -values and source data are provided as a Source Data file.
    Figure Legend Snippet: A Western blotting analyses of the mt-LSU and mt-SSU MRPs and selected assembly factors steady-state levels in GTPBP8-depleted cells. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were resolved via SDS-PAGE and membranes were blotted with antibodies against GTPBP8, mt-LSU MRPs (mL37, uL3m, bL28m, mL49, uL12m, uL15m), mt-SSU MRPs (uS15m, uS16m, uS17m, mS22, mS37) and others (RBFA, GTPBP10, MRM3, TRMT10C, MTG1). SDHA was used as a loading control. B Quantification of MRPs steady-state levels in GTPBP8 knock-out cells compared to control HEK293. Quantification from western blotting ( n = 3 biological replicates) was performed using the Image Lab software and protein signals were normalized to SDHA. Student’s two-tailed t -test was performed. Source data and P -values are provided in a Source Data file. C LFQ mass spectrometry analyses of MitoCarta3.0 mito-pathways in GTPBP8 knock-out cell lines compared to HEK293 ( n = 3 independent experiments). Log 2 (FC) of GTPBP8 KO1 vs HEK293 (red) and GTPBP8 KO2 vs HEK293 (yellow) is represented on the y -axis. Each dot refers to a protein belonging to mt-LSU MRPs, mt-LSU assembly factors, mt-SSU MRPs, mt-SSU assembly factors, mt-RNA processing proteins, mt-transcription and mt-translation pathways. A summary of boxplots is provided in a Supplementary Data File. The complete list of log 2 (FC) values is presented in Supplementary Data . D Sucrose gradient centrifugation analysis to assess mitoribosome sedimentation patterns in GTPBP8 KO1 and GTPBP8 KO2 compared to HEK293 control cells. Mitochondrial lysates were loaded onto 10–30 % isokinetic sucrose gradients and obtained fractions were analyzed via western blotting. Membranes were probed for mt-LSU MRP mL37 and mt-SSU MRP uS15m. Below, quantification using Image Lab software of mL37 and uS15m in the monosome fractions from independent replicates is represented ( n = 3 independent experiments). The samples derived from the same experiment were processed in parallel. The y -axis represents the signal detected in the monosome fraction normalized for the total signal of each sample’s gradient. Data are presented as mean values +/− SD. Student’s two-tailed t -test was performed. P -values and source data are provided as a Source Data file.

    Techniques Used: Western Blot, SDS Page, Control, Knock-Out, Software, Two Tailed Test, Mass Spectrometry, Gradient Centrifugation, Sedimentation, Derivative Assay

    A Western blotting analysis of control HEK293, GTPBP8 KO1 , GTPBP8 KO2 , GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A samples to confirm GTPBP8 knock-out compared to the rescue cell lines. Anti-GTPBP8 antibody was used to compare GTPBP8 endogenous expression with the overexpression in rescue cell lines. SDHA was used as a loading control ( n = 1 independent experiments). B Western blotting analysis to test OxPhos steady-state levels in GTPBP8 KO1 and GTPBP8 KO2 compared to wild-type HEK293 and rescue cell lines GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A . Overexpression was induced prior to the experiment using 1 ng/ml and 50 ng/ml of doxycycline. Whole-cell lysates were resolved via SDS-PAGE. Membranes were probed using the OxPhos antibody cocktail against nuclear-encoded proteins of complexes V (ATP5A) and I (NDUFB8) and mtDNA-encoded complex IV protein MTCO2. SDHA and SDHB were used as loading controls ( n = 1). C Sucrose gradient centrifugation analysis to assess mitoribosome sedimentation patterns in GTPBP8 KO1 and GTPBP8 KO2 compared to HEK293, GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A . Mitochondrial lysates were loaded onto 10-30 % isokinetic sucrose gradients and obtained fractions were analyzed via western blotting. Membranes were probed for mt-LSU MRP, mL37, and mt-SSU MRP, uS15m ( n = 3 independent experiments). D In vivo translation assay of GTPBP8 KO1 and GTPBP8 KO2 cell lines compared to HEK293, GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A . Cells were labeled with a [ 35 S]-methionine and cysteine mix upon cytosolic translation inhibition. Whole cell lysates were resolved by SDS-PAGE and the signal was later detected via autoradiography. The loading was determined using Coomassie staining ( n = 1 independent experiment). Source data are provided as a Source Data file.
    Figure Legend Snippet: A Western blotting analysis of control HEK293, GTPBP8 KO1 , GTPBP8 KO2 , GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A samples to confirm GTPBP8 knock-out compared to the rescue cell lines. Anti-GTPBP8 antibody was used to compare GTPBP8 endogenous expression with the overexpression in rescue cell lines. SDHA was used as a loading control ( n = 1 independent experiments). B Western blotting analysis to test OxPhos steady-state levels in GTPBP8 KO1 and GTPBP8 KO2 compared to wild-type HEK293 and rescue cell lines GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A . Overexpression was induced prior to the experiment using 1 ng/ml and 50 ng/ml of doxycycline. Whole-cell lysates were resolved via SDS-PAGE. Membranes were probed using the OxPhos antibody cocktail against nuclear-encoded proteins of complexes V (ATP5A) and I (NDUFB8) and mtDNA-encoded complex IV protein MTCO2. SDHA and SDHB were used as loading controls ( n = 1). C Sucrose gradient centrifugation analysis to assess mitoribosome sedimentation patterns in GTPBP8 KO1 and GTPBP8 KO2 compared to HEK293, GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A . Mitochondrial lysates were loaded onto 10-30 % isokinetic sucrose gradients and obtained fractions were analyzed via western blotting. Membranes were probed for mt-LSU MRP, mL37, and mt-SSU MRP, uS15m ( n = 3 independent experiments). D In vivo translation assay of GTPBP8 KO1 and GTPBP8 KO2 cell lines compared to HEK293, GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A . Cells were labeled with a [ 35 S]-methionine and cysteine mix upon cytosolic translation inhibition. Whole cell lysates were resolved by SDS-PAGE and the signal was later detected via autoradiography. The loading was determined using Coomassie staining ( n = 1 independent experiment). Source data are provided as a Source Data file.

    Techniques Used: Western Blot, Control, Knock-Out, Expressing, Over Expression, SDS Page, Gradient Centrifugation, Sedimentation, In Vivo, Labeling, Inhibition, Autoradiography, Staining

    human kidney cell line 293  (ATCC)


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    ATCC human kidney cell line 293
    Human Kidney Cell Line 293, 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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    embryonic kidney cell line 293 t  (Thermo Fisher)


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    Thermo Fisher embryonic kidney cell line 293 t
    Embryonic Kidney Cell Line 293 T, supplied by Thermo Fisher, 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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    camp glosensor human embryonic kidney 293 hek293g cell line  (Millipore)

     
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    Millipore camp glosensor human embryonic kidney 293 hek293g cell line
    NanoBRET binding curves for PSB603‐BY630 in <t>HEK293G</t> cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.
    Camp Glosensor Human Embryonic Kidney 293 Hek293g Cell Line, supplied by Millipore, 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 "A novel and selective fluorescent ligand for the study of adenosine A 2B receptors"

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    Journal: Pharmacology Research & Perspectives

    doi: 10.1002/prp2.1223

    NanoBRET binding curves for PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.
    Figure Legend Snippet: NanoBRET binding curves for PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.

    Techniques Used: Binding Assay, Expressing, Incubation

    NanoBRET imaging of PSB603‐BY630 binding to HEK293G cells expressing NLuc‐A 2B R. Cells were incubated with 100 nM PSB603‐BY630 in the absence (A, B) or presence (C, D) of 10 μM MRS‐1706 before addition of furimazine (1:800 dilution) and imaging. Filtered bioluminescence was captured using a 438/24 bandpass filter (cyan A & C). BRET was captured using a 650/50 nm bandpass filter (magenta B & D). Images are representative of those obtained in five independent experiments. Scale bar represents 100 μm.
    Figure Legend Snippet: NanoBRET imaging of PSB603‐BY630 binding to HEK293G cells expressing NLuc‐A 2B R. Cells were incubated with 100 nM PSB603‐BY630 in the absence (A, B) or presence (C, D) of 10 μM MRS‐1706 before addition of furimazine (1:800 dilution) and imaging. Filtered bioluminescence was captured using a 438/24 bandpass filter (cyan A & C). BRET was captured using a 650/50 nm bandpass filter (magenta B & D). Images are representative of those obtained in five independent experiments. Scale bar represents 100 μm.

    Techniques Used: Imaging, Binding Assay, Expressing, Incubation

    NanoBRET competition binding in HEK293G cells exogenously expressing NLuc‐A 2B R. Cells were incubated with 50 nM PSB603‐BY630 in the absence or presence of competing ligands Data are mean ± S.E.M. from five independent experiments. The open and closed bars show the BRET ratio obtained in the absence and presence of 50 nM PSB603‐BY630 respectively.
    Figure Legend Snippet: NanoBRET competition binding in HEK293G cells exogenously expressing NLuc‐A 2B R. Cells were incubated with 50 nM PSB603‐BY630 in the absence or presence of competing ligands Data are mean ± S.E.M. from five independent experiments. The open and closed bars show the BRET ratio obtained in the absence and presence of 50 nM PSB603‐BY630 respectively.

    Techniques Used: Binding Assay, Expressing, Incubation

    Ligand‐binding kinetics of 200 nM PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. BRET ratios for the total binding of 200 nM PSB603‐BY630 were obtained every 30 sec. In parallel, data were also collected in the absence of fluorescent ligand for each time point and these data were subtracted from the total binding to obtain baseline‐corrected values for total binding at each time point. Sixty minutes after addition of 200 nM PSB603‐BY630, 10 μM MRS‐1706 was added to initial dissociation of the fluorescent ligand. Values show mean ± S.E.M of quadruplicate determinations in a single representative experiment. Similar data were obtained in four other experiments. The data points for the dissociation phase of the experiment were then fitted to a single exponential function to determine the the dissociation rate constant ( k off ) in min −1 as described under Methods. In this representative experiment the calculated K off value was 0.056 min −1 . The mean K off value obtained in the five repeat experiments was 0.065 ± 0.003 min ‐1 .
    Figure Legend Snippet: Ligand‐binding kinetics of 200 nM PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. BRET ratios for the total binding of 200 nM PSB603‐BY630 were obtained every 30 sec. In parallel, data were also collected in the absence of fluorescent ligand for each time point and these data were subtracted from the total binding to obtain baseline‐corrected values for total binding at each time point. Sixty minutes after addition of 200 nM PSB603‐BY630, 10 μM MRS‐1706 was added to initial dissociation of the fluorescent ligand. Values show mean ± S.E.M of quadruplicate determinations in a single representative experiment. Similar data were obtained in four other experiments. The data points for the dissociation phase of the experiment were then fitted to a single exponential function to determine the the dissociation rate constant ( k off ) in min −1 as described under Methods. In this representative experiment the calculated K off value was 0.056 min −1 . The mean K off value obtained in the five repeat experiments was 0.065 ± 0.003 min ‐1 .

    Techniques Used: Ligand Binding Assay, Expressing, Binding Assay

    NanoBRET binding curves for PSB603‐BY630 and receptor‐selective fluorescent ligands binding to NLuc‐tagged A 1 , A 2A or A 3 adenosine receptors. (A, B) Total and non‐specific binding of (A) PSB603‐BY630 or (B) EC‐005 to transiently transfected NLuc‐A 2A R obtained in the absence and presence of 1 μM of the A 2A R‐selective antagonist SCH58261. Data are mean ± S.E.M obtained in five independent experiments (each conducted in duplicate). (C, D) Total and non‐specific binding of (C) PSB603‐BY630 or (D) EC‐069 to NLuc‐A 1 R in a stable HEK293T cell line obtained in the absence and presence of 1 μM of the A 1 R‐selective antagonist SLV320. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). (E, F) Total and non‐specific binding of (E) PSB603‐BY630 or (F) AV‐039 to NLuc‐A 3 R in a stable HEK293G cell line obtained in the absence and presence of 1 μM of the A 3 R‐selective antagonist MRS‐1220. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate).
    Figure Legend Snippet: NanoBRET binding curves for PSB603‐BY630 and receptor‐selective fluorescent ligands binding to NLuc‐tagged A 1 , A 2A or A 3 adenosine receptors. (A, B) Total and non‐specific binding of (A) PSB603‐BY630 or (B) EC‐005 to transiently transfected NLuc‐A 2A R obtained in the absence and presence of 1 μM of the A 2A R‐selective antagonist SCH58261. Data are mean ± S.E.M obtained in five independent experiments (each conducted in duplicate). (C, D) Total and non‐specific binding of (C) PSB603‐BY630 or (D) EC‐069 to NLuc‐A 1 R in a stable HEK293T cell line obtained in the absence and presence of 1 μM of the A 1 R‐selective antagonist SLV320. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). (E, F) Total and non‐specific binding of (E) PSB603‐BY630 or (F) AV‐039 to NLuc‐A 3 R in a stable HEK293G cell line obtained in the absence and presence of 1 μM of the A 3 R‐selective antagonist MRS‐1220. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate).

    Techniques Used: Binding Assay, Transfection

    Effect of PSB603‐BY630 on Glosensor cAMP concentration‐response curves to the A 2B ‐selective agonist BAY 60–6583 in (A) HEK293G cells endogenously expressing A 2B R or (B) HEK293G cells overexpressing NLuc‐A 2B R. Concentration response curves were obtained in the absence and presence of 20 nM or 200 nM PSB603‐BY630. Values are mean ± S.E.M. of five separate experiments carried out in triplicate. Data represent peak luminescence response and are expressed as a percentage of the peak luminescence response to 3 μM BAY 60–6583 (in a) or 0.3 μM BAY 60–6583 (in b) obtained in the absence of antagonist in each individual experiment.
    Figure Legend Snippet: Effect of PSB603‐BY630 on Glosensor cAMP concentration‐response curves to the A 2B ‐selective agonist BAY 60–6583 in (A) HEK293G cells endogenously expressing A 2B R or (B) HEK293G cells overexpressing NLuc‐A 2B R. Concentration response curves were obtained in the absence and presence of 20 nM or 200 nM PSB603‐BY630. Values are mean ± S.E.M. of five separate experiments carried out in triplicate. Data represent peak luminescence response and are expressed as a percentage of the peak luminescence response to 3 μM BAY 60–6583 (in a) or 0.3 μM BAY 60–6583 (in b) obtained in the absence of antagonist in each individual experiment.

    Techniques Used: Concentration Assay, Expressing

    Log EC 50 and E MAX values obtained in  HEK293G  cells endogenously expressing A 2B R or  HEK293G  cells expressing recombinant human A 2B R for BAY 60–6593 obtained in the absence and presence of increasing concentrations of PSB603BY630.
    Figure Legend Snippet: Log EC 50 and E MAX values obtained in HEK293G cells endogenously expressing A 2B R or HEK293G cells expressing recombinant human A 2B R for BAY 60–6593 obtained in the absence and presence of increasing concentrations of PSB603BY630.

    Techniques Used: Expressing, Recombinant

    camp glosensor human embryonic kidney 293 hek293g cell line  (Promega)

     
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    Promega camp glosensor human embryonic kidney 293 hek293g cell line
    NanoBRET binding curves for PSB603‐BY630 in <t>HEK293G</t> cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.
    Camp Glosensor Human Embryonic Kidney 293 Hek293g Cell Line, supplied by Promega, 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 "A novel and selective fluorescent ligand for the study of adenosine A 2B receptors"

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    Journal: Pharmacology Research & Perspectives

    doi: 10.1002/prp2.1223

    NanoBRET binding curves for PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.
    Figure Legend Snippet: NanoBRET binding curves for PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.

    Techniques Used: Binding Assay, Expressing, Incubation

    NanoBRET imaging of PSB603‐BY630 binding to HEK293G cells expressing NLuc‐A 2B R. Cells were incubated with 100 nM PSB603‐BY630 in the absence (A, B) or presence (C, D) of 10 μM MRS‐1706 before addition of furimazine (1:800 dilution) and imaging. Filtered bioluminescence was captured using a 438/24 bandpass filter (cyan A & C). BRET was captured using a 650/50 nm bandpass filter (magenta B & D). Images are representative of those obtained in five independent experiments. Scale bar represents 100 μm.
    Figure Legend Snippet: NanoBRET imaging of PSB603‐BY630 binding to HEK293G cells expressing NLuc‐A 2B R. Cells were incubated with 100 nM PSB603‐BY630 in the absence (A, B) or presence (C, D) of 10 μM MRS‐1706 before addition of furimazine (1:800 dilution) and imaging. Filtered bioluminescence was captured using a 438/24 bandpass filter (cyan A & C). BRET was captured using a 650/50 nm bandpass filter (magenta B & D). Images are representative of those obtained in five independent experiments. Scale bar represents 100 μm.

    Techniques Used: Imaging, Binding Assay, Expressing, Incubation

    NanoBRET competition binding in HEK293G cells exogenously expressing NLuc‐A 2B R. Cells were incubated with 50 nM PSB603‐BY630 in the absence or presence of competing ligands Data are mean ± S.E.M. from five independent experiments. The open and closed bars show the BRET ratio obtained in the absence and presence of 50 nM PSB603‐BY630 respectively.
    Figure Legend Snippet: NanoBRET competition binding in HEK293G cells exogenously expressing NLuc‐A 2B R. Cells were incubated with 50 nM PSB603‐BY630 in the absence or presence of competing ligands Data are mean ± S.E.M. from five independent experiments. The open and closed bars show the BRET ratio obtained in the absence and presence of 50 nM PSB603‐BY630 respectively.

    Techniques Used: Binding Assay, Expressing, Incubation

    Ligand‐binding kinetics of 200 nM PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. BRET ratios for the total binding of 200 nM PSB603‐BY630 were obtained every 30 sec. In parallel, data were also collected in the absence of fluorescent ligand for each time point and these data were subtracted from the total binding to obtain baseline‐corrected values for total binding at each time point. Sixty minutes after addition of 200 nM PSB603‐BY630, 10 μM MRS‐1706 was added to initial dissociation of the fluorescent ligand. Values show mean ± S.E.M of quadruplicate determinations in a single representative experiment. Similar data were obtained in four other experiments. The data points for the dissociation phase of the experiment were then fitted to a single exponential function to determine the the dissociation rate constant ( k off ) in min −1 as described under Methods. In this representative experiment the calculated K off value was 0.056 min −1 . The mean K off value obtained in the five repeat experiments was 0.065 ± 0.003 min ‐1 .
    Figure Legend Snippet: Ligand‐binding kinetics of 200 nM PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. BRET ratios for the total binding of 200 nM PSB603‐BY630 were obtained every 30 sec. In parallel, data were also collected in the absence of fluorescent ligand for each time point and these data were subtracted from the total binding to obtain baseline‐corrected values for total binding at each time point. Sixty minutes after addition of 200 nM PSB603‐BY630, 10 μM MRS‐1706 was added to initial dissociation of the fluorescent ligand. Values show mean ± S.E.M of quadruplicate determinations in a single representative experiment. Similar data were obtained in four other experiments. The data points for the dissociation phase of the experiment were then fitted to a single exponential function to determine the the dissociation rate constant ( k off ) in min −1 as described under Methods. In this representative experiment the calculated K off value was 0.056 min −1 . The mean K off value obtained in the five repeat experiments was 0.065 ± 0.003 min ‐1 .

    Techniques Used: Ligand Binding Assay, Expressing, Binding Assay

    NanoBRET binding curves for PSB603‐BY630 and receptor‐selective fluorescent ligands binding to NLuc‐tagged A 1 , A 2A or A 3 adenosine receptors. (A, B) Total and non‐specific binding of (A) PSB603‐BY630 or (B) EC‐005 to transiently transfected NLuc‐A 2A R obtained in the absence and presence of 1 μM of the A 2A R‐selective antagonist SCH58261. Data are mean ± S.E.M obtained in five independent experiments (each conducted in duplicate). (C, D) Total and non‐specific binding of (C) PSB603‐BY630 or (D) EC‐069 to NLuc‐A 1 R in a stable HEK293T cell line obtained in the absence and presence of 1 μM of the A 1 R‐selective antagonist SLV320. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). (E, F) Total and non‐specific binding of (E) PSB603‐BY630 or (F) AV‐039 to NLuc‐A 3 R in a stable HEK293G cell line obtained in the absence and presence of 1 μM of the A 3 R‐selective antagonist MRS‐1220. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate).
    Figure Legend Snippet: NanoBRET binding curves for PSB603‐BY630 and receptor‐selective fluorescent ligands binding to NLuc‐tagged A 1 , A 2A or A 3 adenosine receptors. (A, B) Total and non‐specific binding of (A) PSB603‐BY630 or (B) EC‐005 to transiently transfected NLuc‐A 2A R obtained in the absence and presence of 1 μM of the A 2A R‐selective antagonist SCH58261. Data are mean ± S.E.M obtained in five independent experiments (each conducted in duplicate). (C, D) Total and non‐specific binding of (C) PSB603‐BY630 or (D) EC‐069 to NLuc‐A 1 R in a stable HEK293T cell line obtained in the absence and presence of 1 μM of the A 1 R‐selective antagonist SLV320. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). (E, F) Total and non‐specific binding of (E) PSB603‐BY630 or (F) AV‐039 to NLuc‐A 3 R in a stable HEK293G cell line obtained in the absence and presence of 1 μM of the A 3 R‐selective antagonist MRS‐1220. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate).

    Techniques Used: Binding Assay, Transfection

    Effect of PSB603‐BY630 on Glosensor cAMP concentration‐response curves to the A 2B ‐selective agonist BAY 60–6583 in (A) HEK293G cells endogenously expressing A 2B R or (B) HEK293G cells overexpressing NLuc‐A 2B R. Concentration response curves were obtained in the absence and presence of 20 nM or 200 nM PSB603‐BY630. Values are mean ± S.E.M. of five separate experiments carried out in triplicate. Data represent peak luminescence response and are expressed as a percentage of the peak luminescence response to 3 μM BAY 60–6583 (in a) or 0.3 μM BAY 60–6583 (in b) obtained in the absence of antagonist in each individual experiment.
    Figure Legend Snippet: Effect of PSB603‐BY630 on Glosensor cAMP concentration‐response curves to the A 2B ‐selective agonist BAY 60–6583 in (A) HEK293G cells endogenously expressing A 2B R or (B) HEK293G cells overexpressing NLuc‐A 2B R. Concentration response curves were obtained in the absence and presence of 20 nM or 200 nM PSB603‐BY630. Values are mean ± S.E.M. of five separate experiments carried out in triplicate. Data represent peak luminescence response and are expressed as a percentage of the peak luminescence response to 3 μM BAY 60–6583 (in a) or 0.3 μM BAY 60–6583 (in b) obtained in the absence of antagonist in each individual experiment.

    Techniques Used: Concentration Assay, Expressing

    Log EC 50 and E MAX values obtained in  HEK293G  cells endogenously expressing A 2B R or  HEK293G  cells expressing recombinant human A 2B R for BAY 60–6593 obtained in the absence and presence of increasing concentrations of PSB603BY630.
    Figure Legend Snippet: Log EC 50 and E MAX values obtained in HEK293G cells endogenously expressing A 2B R or HEK293G cells expressing recombinant human A 2B R for BAY 60–6593 obtained in the absence and presence of increasing concentrations of PSB603BY630.

    Techniques Used: Expressing, Recombinant

    human embryonic kidney cell line hek 293  (ATCC)


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    ATCC human embryonic kidney cell line hek 293
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    human embryonic kidney cell line hek 293  (ATCC)


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    t rex human embryonic kidney 293 hek293ji cell line  (ATCC)


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    ATCC hek 293 a human embryonic kidney cell line
    Phosphorylation-independent MAb production and epitope mapping. (A) Reactivity of MAbs with TDP-43-Flag recombinant protein of about 46 kDa (lane 1) overexpressed in <t>HEK</t> <t>293</t> cells by Western blot. Anti-Flag MAb (Flag) was used as the positive control antibody. Leucine-rich repeat-containing protein 15 (LRRC15)-His fusion protein (lane 2) served as the negative control, which was revealed by anti-His MAb (HIS). (B) Schematic representation of the TDP-43 fragments employed for epitope mapping. (C) Mapping of TDP-43 domains with epitopes recognized by the 10 generated MAbs. This mapping was performed across the entire length of TDP-43 using immunoblot and indirect peptide ELISA. RRM, RNA-recognition motif; NLS, bipartite nuclear localization signal; NES, nuclear export signal.
    Hek 293 A Human Embryonic Kidney Cell Line, 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 embryonic kidney cell line 293 t
    Phosphorylation-independent MAb production and epitope mapping. (A) Reactivity of MAbs with TDP-43-Flag recombinant protein of about 46 kDa (lane 1) overexpressed in <t>HEK</t> <t>293</t> cells by Western blot. Anti-Flag MAb (Flag) was used as the positive control antibody. Leucine-rich repeat-containing protein 15 (LRRC15)-His fusion protein (lane 2) served as the negative control, which was revealed by anti-His MAb (HIS). (B) Schematic representation of the TDP-43 fragments employed for epitope mapping. (C) Mapping of TDP-43 domains with epitopes recognized by the 10 generated MAbs. This mapping was performed across the entire length of TDP-43 using immunoblot and indirect peptide ELISA. RRM, RNA-recognition motif; NLS, bipartite nuclear localization signal; NES, nuclear export signal.
    Embryonic Kidney Cell Line 293 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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    Thermo Fisher flp in t rex human embryonic kidney 293 hek293 cell line
    A Western blotting to confirm GTPBP8 knock-out. Whole-cell lysates from <t>HEK293,</t> GTPBP8 KO1 and GTPBP8 KO2 were used for SDS-PAGE analysis. GTPBP8 antibody was used to detect GTPBP8 endogenous levels; SDHA antibody was used as a loading control. B Growth rates of GTPBP8 knock-out cells in galactose medium. HEK293 (black), GTPBP8 KO1 (red), and GTPBP8 KO2 (yellow) were cultured in DMEM containing 0.9 g/l galactose ( n = 3 independent experiments). The mean average cell number per each time point is indicated, and error bars = ± 1 standard deviation (SD). Statistical analyses were performed using the Student’s two-tailed t -test. P -values and source data are provided in the Source Data file. C Western blotting to assess OxPhos complexes steady-state levels in GTPBP8 knock-out cell lines. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were resolved via SDS-PAGE. Membranes were probed using the OxPhos antibody cocktail against nuclear-encoded proteins of complexes V (ATP5A) and I (NDUFB8) and mtDNA-encoded complex IV protein MTCO2. GAPDH was used as a loading control. D LFQ mass spectrometry analyses of OxPhos complexes in GTPBP8 knock-out cell lines compared to HEK293 ( n = 3 independent experiments). Log 2 (FC) of GTPBP8 KO1 vs HEK293 (red) and GTPBP8 KO2 vs HEK293 (yellow) is represented on the y -axis for each OxPhos complex. Each dot refers to a protein, which constitutes the specific complex. A summary of boxplots is provided in a Source Data file. All the log 2 (FC) values are listed in Supplementary Data . E Assessment of OxPhos complex I and complex IV activities in GTPBP8 knock-out cell lines compared to control. Mitochondrial lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 cell lines were resolved by BN-PAGE and stained using complex I (CI) and complex IV (CIV) -specific substrates ( n = 1 independent experiment). Coomassie staining was used as a loading control. SC supercomplexes. Source data are provided as a Source Data file.
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    ATCC human kidney cell line 293
    A Western blotting to confirm GTPBP8 knock-out. Whole-cell lysates from <t>HEK293,</t> GTPBP8 KO1 and GTPBP8 KO2 were used for SDS-PAGE analysis. GTPBP8 antibody was used to detect GTPBP8 endogenous levels; SDHA antibody was used as a loading control. B Growth rates of GTPBP8 knock-out cells in galactose medium. HEK293 (black), GTPBP8 KO1 (red), and GTPBP8 KO2 (yellow) were cultured in DMEM containing 0.9 g/l galactose ( n = 3 independent experiments). The mean average cell number per each time point is indicated, and error bars = ± 1 standard deviation (SD). Statistical analyses were performed using the Student’s two-tailed t -test. P -values and source data are provided in the Source Data file. C Western blotting to assess OxPhos complexes steady-state levels in GTPBP8 knock-out cell lines. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were resolved via SDS-PAGE. Membranes were probed using the OxPhos antibody cocktail against nuclear-encoded proteins of complexes V (ATP5A) and I (NDUFB8) and mtDNA-encoded complex IV protein MTCO2. GAPDH was used as a loading control. D LFQ mass spectrometry analyses of OxPhos complexes in GTPBP8 knock-out cell lines compared to HEK293 ( n = 3 independent experiments). Log 2 (FC) of GTPBP8 KO1 vs HEK293 (red) and GTPBP8 KO2 vs HEK293 (yellow) is represented on the y -axis for each OxPhos complex. Each dot refers to a protein, which constitutes the specific complex. A summary of boxplots is provided in a Source Data file. All the log 2 (FC) values are listed in Supplementary Data . E Assessment of OxPhos complex I and complex IV activities in GTPBP8 knock-out cell lines compared to control. Mitochondrial lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 cell lines were resolved by BN-PAGE and stained using complex I (CI) and complex IV (CIV) -specific substrates ( n = 1 independent experiment). Coomassie staining was used as a loading control. SC supercomplexes. Source data are provided as a Source Data file.
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    Thermo Fisher embryonic kidney cell line 293 t
    A Western blotting to confirm GTPBP8 knock-out. Whole-cell lysates from <t>HEK293,</t> GTPBP8 KO1 and GTPBP8 KO2 were used for SDS-PAGE analysis. GTPBP8 antibody was used to detect GTPBP8 endogenous levels; SDHA antibody was used as a loading control. B Growth rates of GTPBP8 knock-out cells in galactose medium. HEK293 (black), GTPBP8 KO1 (red), and GTPBP8 KO2 (yellow) were cultured in DMEM containing 0.9 g/l galactose ( n = 3 independent experiments). The mean average cell number per each time point is indicated, and error bars = ± 1 standard deviation (SD). Statistical analyses were performed using the Student’s two-tailed t -test. P -values and source data are provided in the Source Data file. C Western blotting to assess OxPhos complexes steady-state levels in GTPBP8 knock-out cell lines. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were resolved via SDS-PAGE. Membranes were probed using the OxPhos antibody cocktail against nuclear-encoded proteins of complexes V (ATP5A) and I (NDUFB8) and mtDNA-encoded complex IV protein MTCO2. GAPDH was used as a loading control. D LFQ mass spectrometry analyses of OxPhos complexes in GTPBP8 knock-out cell lines compared to HEK293 ( n = 3 independent experiments). Log 2 (FC) of GTPBP8 KO1 vs HEK293 (red) and GTPBP8 KO2 vs HEK293 (yellow) is represented on the y -axis for each OxPhos complex. Each dot refers to a protein, which constitutes the specific complex. A summary of boxplots is provided in a Source Data file. All the log 2 (FC) values are listed in Supplementary Data . E Assessment of OxPhos complex I and complex IV activities in GTPBP8 knock-out cell lines compared to control. Mitochondrial lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 cell lines were resolved by BN-PAGE and stained using complex I (CI) and complex IV (CIV) -specific substrates ( n = 1 independent experiment). Coomassie staining was used as a loading control. SC supercomplexes. Source data are provided as a Source Data file.
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    Millipore camp glosensor human embryonic kidney 293 hek293g cell line
    NanoBRET binding curves for PSB603‐BY630 in <t>HEK293G</t> cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.
    Camp Glosensor Human Embryonic Kidney 293 Hek293g Cell Line, supplied by Millipore, 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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    Promega camp glosensor human embryonic kidney 293 hek293g cell line
    NanoBRET binding curves for PSB603‐BY630 in <t>HEK293G</t> cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.
    Camp Glosensor Human Embryonic Kidney 293 Hek293g Cell Line, supplied by Promega, 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 human embryonic kidney cell line hek 293
    NanoBRET binding curves for PSB603‐BY630 in <t>HEK293G</t> cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.
    Human Embryonic Kidney Cell Line Hek 293, 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 t rex human embryonic kidney 293 hek293ji cell line
    NanoBRET binding curves for PSB603‐BY630 in <t>HEK293G</t> cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.
    T Rex Human Embryonic Kidney 293 Hek293ji Cell Line, 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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    Image Search Results


    Phosphorylation-independent MAb production and epitope mapping. (A) Reactivity of MAbs with TDP-43-Flag recombinant protein of about 46 kDa (lane 1) overexpressed in HEK 293 cells by Western blot. Anti-Flag MAb (Flag) was used as the positive control antibody. Leucine-rich repeat-containing protein 15 (LRRC15)-His fusion protein (lane 2) served as the negative control, which was revealed by anti-His MAb (HIS). (B) Schematic representation of the TDP-43 fragments employed for epitope mapping. (C) Mapping of TDP-43 domains with epitopes recognized by the 10 generated MAbs. This mapping was performed across the entire length of TDP-43 using immunoblot and indirect peptide ELISA. RRM, RNA-recognition motif; NLS, bipartite nuclear localization signal; NES, nuclear export signal.

    Journal: Journal of Neuropathology and Experimental Neurology

    Article Title: Production and characterization of novel monoclonal antibodies against pathological human TDP-43 proteins

    doi: 10.1093/jnen/nlae042

    Figure Lengend Snippet: Phosphorylation-independent MAb production and epitope mapping. (A) Reactivity of MAbs with TDP-43-Flag recombinant protein of about 46 kDa (lane 1) overexpressed in HEK 293 cells by Western blot. Anti-Flag MAb (Flag) was used as the positive control antibody. Leucine-rich repeat-containing protein 15 (LRRC15)-His fusion protein (lane 2) served as the negative control, which was revealed by anti-His MAb (HIS). (B) Schematic representation of the TDP-43 fragments employed for epitope mapping. (C) Mapping of TDP-43 domains with epitopes recognized by the 10 generated MAbs. This mapping was performed across the entire length of TDP-43 using immunoblot and indirect peptide ELISA. RRM, RNA-recognition motif; NLS, bipartite nuclear localization signal; NES, nuclear export signal.

    Article Snippet: Two cell lines, SH-SY5Y (a human neuroblastoma cell line) and HEK 293 (a human embryonic kidney cell line), were sourced from the American Type Culture Collection (ATCC, Manassas, VA).

    Techniques: Recombinant, Western Blot, Positive Control, Negative Control, Generated, Peptide ELISA

    A Western blotting to confirm GTPBP8 knock-out. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were used for SDS-PAGE analysis. GTPBP8 antibody was used to detect GTPBP8 endogenous levels; SDHA antibody was used as a loading control. B Growth rates of GTPBP8 knock-out cells in galactose medium. HEK293 (black), GTPBP8 KO1 (red), and GTPBP8 KO2 (yellow) were cultured in DMEM containing 0.9 g/l galactose ( n = 3 independent experiments). The mean average cell number per each time point is indicated, and error bars = ± 1 standard deviation (SD). Statistical analyses were performed using the Student’s two-tailed t -test. P -values and source data are provided in the Source Data file. C Western blotting to assess OxPhos complexes steady-state levels in GTPBP8 knock-out cell lines. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were resolved via SDS-PAGE. Membranes were probed using the OxPhos antibody cocktail against nuclear-encoded proteins of complexes V (ATP5A) and I (NDUFB8) and mtDNA-encoded complex IV protein MTCO2. GAPDH was used as a loading control. D LFQ mass spectrometry analyses of OxPhos complexes in GTPBP8 knock-out cell lines compared to HEK293 ( n = 3 independent experiments). Log 2 (FC) of GTPBP8 KO1 vs HEK293 (red) and GTPBP8 KO2 vs HEK293 (yellow) is represented on the y -axis for each OxPhos complex. Each dot refers to a protein, which constitutes the specific complex. A summary of boxplots is provided in a Source Data file. All the log 2 (FC) values are listed in Supplementary Data . E Assessment of OxPhos complex I and complex IV activities in GTPBP8 knock-out cell lines compared to control. Mitochondrial lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 cell lines were resolved by BN-PAGE and stained using complex I (CI) and complex IV (CIV) -specific substrates ( n = 1 independent experiment). Coomassie staining was used as a loading control. SC supercomplexes. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: GTPBP8 plays a role in mitoribosome formation in human mitochondria

    doi: 10.1038/s41467-024-50011-x

    Figure Lengend Snippet: A Western blotting to confirm GTPBP8 knock-out. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were used for SDS-PAGE analysis. GTPBP8 antibody was used to detect GTPBP8 endogenous levels; SDHA antibody was used as a loading control. B Growth rates of GTPBP8 knock-out cells in galactose medium. HEK293 (black), GTPBP8 KO1 (red), and GTPBP8 KO2 (yellow) were cultured in DMEM containing 0.9 g/l galactose ( n = 3 independent experiments). The mean average cell number per each time point is indicated, and error bars = ± 1 standard deviation (SD). Statistical analyses were performed using the Student’s two-tailed t -test. P -values and source data are provided in the Source Data file. C Western blotting to assess OxPhos complexes steady-state levels in GTPBP8 knock-out cell lines. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were resolved via SDS-PAGE. Membranes were probed using the OxPhos antibody cocktail against nuclear-encoded proteins of complexes V (ATP5A) and I (NDUFB8) and mtDNA-encoded complex IV protein MTCO2. GAPDH was used as a loading control. D LFQ mass spectrometry analyses of OxPhos complexes in GTPBP8 knock-out cell lines compared to HEK293 ( n = 3 independent experiments). Log 2 (FC) of GTPBP8 KO1 vs HEK293 (red) and GTPBP8 KO2 vs HEK293 (yellow) is represented on the y -axis for each OxPhos complex. Each dot refers to a protein, which constitutes the specific complex. A summary of boxplots is provided in a Source Data file. All the log 2 (FC) values are listed in Supplementary Data . E Assessment of OxPhos complex I and complex IV activities in GTPBP8 knock-out cell lines compared to control. Mitochondrial lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 cell lines were resolved by BN-PAGE and stained using complex I (CI) and complex IV (CIV) -specific substrates ( n = 1 independent experiment). Coomassie staining was used as a loading control. SC supercomplexes. Source data are provided as a Source Data file.

    Article Snippet: Stable mammalian cell lines that enable doxycycline-inducible expression of C-terminally FLAG-tagged GTPBP8 (GTPBP8::FLAG) were generated using the Flp-In T-Rex human embryonic kidney 293 (HEK293) cell line (Invitrogen).

    Techniques: Western Blot, Knock-Out, SDS Page, Control, Cell Culture, Standard Deviation, Two Tailed Test, Mass Spectrometry, Staining

    A In vivo translation assay of GTPBP8 KO1 and GTPBP8 KO2 cell lines compared to HEK293. Cells were labeled with a [ 35 S]-methionine and cysteine mix upon cytosolic translation inhibition. Whole cell lysates were resolved by SDS-PAGE and the signal was later detected via autoradiography. Loading was determined using Coomassie staining ( n = 3 independent experiments). B Mitoribosome profiling analysis of GTPBP8 KO1 compared to HEK293. The ratio of the mitoribosome-protected fragments (RPFs) in GTPBP8 KO1 vs control for each mitochondrial transcript is represented on the y -axis. The data were determined via MitoRiboSeq and refer to a single experiment. C Quantitative real-time PCR of mt-mRNAs and mt-rRNAs steady-state levels in HEK293 cells depleted of GTPBP8 compared to control. Total RNA isolated from HEK293, GTPBP8 KO1 and GTPBP8 KO2 was used ( n = 3 biological replicates, data presented as mean values ± 1 SD). Student’s two-tailed t -test was performed. P -values and source data are provided in a Source Data file. The significance cut-off was set at p < 0.05, with * p < 0.05, ** p < 0.01, and *** p < 0.001. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: GTPBP8 plays a role in mitoribosome formation in human mitochondria

    doi: 10.1038/s41467-024-50011-x

    Figure Lengend Snippet: A In vivo translation assay of GTPBP8 KO1 and GTPBP8 KO2 cell lines compared to HEK293. Cells were labeled with a [ 35 S]-methionine and cysteine mix upon cytosolic translation inhibition. Whole cell lysates were resolved by SDS-PAGE and the signal was later detected via autoradiography. Loading was determined using Coomassie staining ( n = 3 independent experiments). B Mitoribosome profiling analysis of GTPBP8 KO1 compared to HEK293. The ratio of the mitoribosome-protected fragments (RPFs) in GTPBP8 KO1 vs control for each mitochondrial transcript is represented on the y -axis. The data were determined via MitoRiboSeq and refer to a single experiment. C Quantitative real-time PCR of mt-mRNAs and mt-rRNAs steady-state levels in HEK293 cells depleted of GTPBP8 compared to control. Total RNA isolated from HEK293, GTPBP8 KO1 and GTPBP8 KO2 was used ( n = 3 biological replicates, data presented as mean values ± 1 SD). Student’s two-tailed t -test was performed. P -values and source data are provided in a Source Data file. The significance cut-off was set at p < 0.05, with * p < 0.05, ** p < 0.01, and *** p < 0.001. Source data are provided as a Source Data file.

    Article Snippet: Stable mammalian cell lines that enable doxycycline-inducible expression of C-terminally FLAG-tagged GTPBP8 (GTPBP8::FLAG) were generated using the Flp-In T-Rex human embryonic kidney 293 (HEK293) cell line (Invitrogen).

    Techniques: In Vivo, Labeling, Inhibition, SDS Page, Autoradiography, Staining, Control, Real-time Polymerase Chain Reaction, Isolation, Two Tailed Test

    A Western blotting analyses of the mt-LSU and mt-SSU MRPs and selected assembly factors steady-state levels in GTPBP8-depleted cells. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were resolved via SDS-PAGE and membranes were blotted with antibodies against GTPBP8, mt-LSU MRPs (mL37, uL3m, bL28m, mL49, uL12m, uL15m), mt-SSU MRPs (uS15m, uS16m, uS17m, mS22, mS37) and others (RBFA, GTPBP10, MRM3, TRMT10C, MTG1). SDHA was used as a loading control. B Quantification of MRPs steady-state levels in GTPBP8 knock-out cells compared to control HEK293. Quantification from western blotting ( n = 3 biological replicates) was performed using the Image Lab software and protein signals were normalized to SDHA. Student’s two-tailed t -test was performed. Source data and P -values are provided in a Source Data file. C LFQ mass spectrometry analyses of MitoCarta3.0 mito-pathways in GTPBP8 knock-out cell lines compared to HEK293 ( n = 3 independent experiments). Log 2 (FC) of GTPBP8 KO1 vs HEK293 (red) and GTPBP8 KO2 vs HEK293 (yellow) is represented on the y -axis. Each dot refers to a protein belonging to mt-LSU MRPs, mt-LSU assembly factors, mt-SSU MRPs, mt-SSU assembly factors, mt-RNA processing proteins, mt-transcription and mt-translation pathways. A summary of boxplots is provided in a Supplementary Data File. The complete list of log 2 (FC) values is presented in Supplementary Data . D Sucrose gradient centrifugation analysis to assess mitoribosome sedimentation patterns in GTPBP8 KO1 and GTPBP8 KO2 compared to HEK293 control cells. Mitochondrial lysates were loaded onto 10–30 % isokinetic sucrose gradients and obtained fractions were analyzed via western blotting. Membranes were probed for mt-LSU MRP mL37 and mt-SSU MRP uS15m. Below, quantification using Image Lab software of mL37 and uS15m in the monosome fractions from independent replicates is represented ( n = 3 independent experiments). The samples derived from the same experiment were processed in parallel. The y -axis represents the signal detected in the monosome fraction normalized for the total signal of each sample’s gradient. Data are presented as mean values +/− SD. Student’s two-tailed t -test was performed. P -values and source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: GTPBP8 plays a role in mitoribosome formation in human mitochondria

    doi: 10.1038/s41467-024-50011-x

    Figure Lengend Snippet: A Western blotting analyses of the mt-LSU and mt-SSU MRPs and selected assembly factors steady-state levels in GTPBP8-depleted cells. Whole-cell lysates from HEK293, GTPBP8 KO1 and GTPBP8 KO2 were resolved via SDS-PAGE and membranes were blotted with antibodies against GTPBP8, mt-LSU MRPs (mL37, uL3m, bL28m, mL49, uL12m, uL15m), mt-SSU MRPs (uS15m, uS16m, uS17m, mS22, mS37) and others (RBFA, GTPBP10, MRM3, TRMT10C, MTG1). SDHA was used as a loading control. B Quantification of MRPs steady-state levels in GTPBP8 knock-out cells compared to control HEK293. Quantification from western blotting ( n = 3 biological replicates) was performed using the Image Lab software and protein signals were normalized to SDHA. Student’s two-tailed t -test was performed. Source data and P -values are provided in a Source Data file. C LFQ mass spectrometry analyses of MitoCarta3.0 mito-pathways in GTPBP8 knock-out cell lines compared to HEK293 ( n = 3 independent experiments). Log 2 (FC) of GTPBP8 KO1 vs HEK293 (red) and GTPBP8 KO2 vs HEK293 (yellow) is represented on the y -axis. Each dot refers to a protein belonging to mt-LSU MRPs, mt-LSU assembly factors, mt-SSU MRPs, mt-SSU assembly factors, mt-RNA processing proteins, mt-transcription and mt-translation pathways. A summary of boxplots is provided in a Supplementary Data File. The complete list of log 2 (FC) values is presented in Supplementary Data . D Sucrose gradient centrifugation analysis to assess mitoribosome sedimentation patterns in GTPBP8 KO1 and GTPBP8 KO2 compared to HEK293 control cells. Mitochondrial lysates were loaded onto 10–30 % isokinetic sucrose gradients and obtained fractions were analyzed via western blotting. Membranes were probed for mt-LSU MRP mL37 and mt-SSU MRP uS15m. Below, quantification using Image Lab software of mL37 and uS15m in the monosome fractions from independent replicates is represented ( n = 3 independent experiments). The samples derived from the same experiment were processed in parallel. The y -axis represents the signal detected in the monosome fraction normalized for the total signal of each sample’s gradient. Data are presented as mean values +/− SD. Student’s two-tailed t -test was performed. P -values and source data are provided as a Source Data file.

    Article Snippet: Stable mammalian cell lines that enable doxycycline-inducible expression of C-terminally FLAG-tagged GTPBP8 (GTPBP8::FLAG) were generated using the Flp-In T-Rex human embryonic kidney 293 (HEK293) cell line (Invitrogen).

    Techniques: Western Blot, SDS Page, Control, Knock-Out, Software, Two Tailed Test, Mass Spectrometry, Gradient Centrifugation, Sedimentation, Derivative Assay

    A Western blotting analysis of control HEK293, GTPBP8 KO1 , GTPBP8 KO2 , GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A samples to confirm GTPBP8 knock-out compared to the rescue cell lines. Anti-GTPBP8 antibody was used to compare GTPBP8 endogenous expression with the overexpression in rescue cell lines. SDHA was used as a loading control ( n = 1 independent experiments). B Western blotting analysis to test OxPhos steady-state levels in GTPBP8 KO1 and GTPBP8 KO2 compared to wild-type HEK293 and rescue cell lines GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A . Overexpression was induced prior to the experiment using 1 ng/ml and 50 ng/ml of doxycycline. Whole-cell lysates were resolved via SDS-PAGE. Membranes were probed using the OxPhos antibody cocktail against nuclear-encoded proteins of complexes V (ATP5A) and I (NDUFB8) and mtDNA-encoded complex IV protein MTCO2. SDHA and SDHB were used as loading controls ( n = 1). C Sucrose gradient centrifugation analysis to assess mitoribosome sedimentation patterns in GTPBP8 KO1 and GTPBP8 KO2 compared to HEK293, GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A . Mitochondrial lysates were loaded onto 10-30 % isokinetic sucrose gradients and obtained fractions were analyzed via western blotting. Membranes were probed for mt-LSU MRP, mL37, and mt-SSU MRP, uS15m ( n = 3 independent experiments). D In vivo translation assay of GTPBP8 KO1 and GTPBP8 KO2 cell lines compared to HEK293, GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A . Cells were labeled with a [ 35 S]-methionine and cysteine mix upon cytosolic translation inhibition. Whole cell lysates were resolved by SDS-PAGE and the signal was later detected via autoradiography. The loading was determined using Coomassie staining ( n = 1 independent experiment). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: GTPBP8 plays a role in mitoribosome formation in human mitochondria

    doi: 10.1038/s41467-024-50011-x

    Figure Lengend Snippet: A Western blotting analysis of control HEK293, GTPBP8 KO1 , GTPBP8 KO2 , GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A samples to confirm GTPBP8 knock-out compared to the rescue cell lines. Anti-GTPBP8 antibody was used to compare GTPBP8 endogenous expression with the overexpression in rescue cell lines. SDHA was used as a loading control ( n = 1 independent experiments). B Western blotting analysis to test OxPhos steady-state levels in GTPBP8 KO1 and GTPBP8 KO2 compared to wild-type HEK293 and rescue cell lines GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A . Overexpression was induced prior to the experiment using 1 ng/ml and 50 ng/ml of doxycycline. Whole-cell lysates were resolved via SDS-PAGE. Membranes were probed using the OxPhos antibody cocktail against nuclear-encoded proteins of complexes V (ATP5A) and I (NDUFB8) and mtDNA-encoded complex IV protein MTCO2. SDHA and SDHB were used as loading controls ( n = 1). C Sucrose gradient centrifugation analysis to assess mitoribosome sedimentation patterns in GTPBP8 KO1 and GTPBP8 KO2 compared to HEK293, GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A . Mitochondrial lysates were loaded onto 10-30 % isokinetic sucrose gradients and obtained fractions were analyzed via western blotting. Membranes were probed for mt-LSU MRP, mL37, and mt-SSU MRP, uS15m ( n = 3 independent experiments). D In vivo translation assay of GTPBP8 KO1 and GTPBP8 KO2 cell lines compared to HEK293, GTPBP8 RESCUE_wt and GTPBP8 RESCUE_S124A . Cells were labeled with a [ 35 S]-methionine and cysteine mix upon cytosolic translation inhibition. Whole cell lysates were resolved by SDS-PAGE and the signal was later detected via autoradiography. The loading was determined using Coomassie staining ( n = 1 independent experiment). Source data are provided as a Source Data file.

    Article Snippet: Stable mammalian cell lines that enable doxycycline-inducible expression of C-terminally FLAG-tagged GTPBP8 (GTPBP8::FLAG) were generated using the Flp-In T-Rex human embryonic kidney 293 (HEK293) cell line (Invitrogen).

    Techniques: Western Blot, Control, Knock-Out, Expressing, Over Expression, SDS Page, Gradient Centrifugation, Sedimentation, In Vivo, Labeling, Inhibition, Autoradiography, Staining

    NanoBRET binding curves for PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: NanoBRET binding curves for PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Binding Assay, Expressing, Incubation

    NanoBRET imaging of PSB603‐BY630 binding to HEK293G cells expressing NLuc‐A 2B R. Cells were incubated with 100 nM PSB603‐BY630 in the absence (A, B) or presence (C, D) of 10 μM MRS‐1706 before addition of furimazine (1:800 dilution) and imaging. Filtered bioluminescence was captured using a 438/24 bandpass filter (cyan A & C). BRET was captured using a 650/50 nm bandpass filter (magenta B & D). Images are representative of those obtained in five independent experiments. Scale bar represents 100 μm.

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: NanoBRET imaging of PSB603‐BY630 binding to HEK293G cells expressing NLuc‐A 2B R. Cells were incubated with 100 nM PSB603‐BY630 in the absence (A, B) or presence (C, D) of 10 μM MRS‐1706 before addition of furimazine (1:800 dilution) and imaging. Filtered bioluminescence was captured using a 438/24 bandpass filter (cyan A & C). BRET was captured using a 650/50 nm bandpass filter (magenta B & D). Images are representative of those obtained in five independent experiments. Scale bar represents 100 μm.

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Imaging, Binding Assay, Expressing, Incubation

    NanoBRET competition binding in HEK293G cells exogenously expressing NLuc‐A 2B R. Cells were incubated with 50 nM PSB603‐BY630 in the absence or presence of competing ligands Data are mean ± S.E.M. from five independent experiments. The open and closed bars show the BRET ratio obtained in the absence and presence of 50 nM PSB603‐BY630 respectively.

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: NanoBRET competition binding in HEK293G cells exogenously expressing NLuc‐A 2B R. Cells were incubated with 50 nM PSB603‐BY630 in the absence or presence of competing ligands Data are mean ± S.E.M. from five independent experiments. The open and closed bars show the BRET ratio obtained in the absence and presence of 50 nM PSB603‐BY630 respectively.

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Binding Assay, Expressing, Incubation

    Ligand‐binding kinetics of 200 nM PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. BRET ratios for the total binding of 200 nM PSB603‐BY630 were obtained every 30 sec. In parallel, data were also collected in the absence of fluorescent ligand for each time point and these data were subtracted from the total binding to obtain baseline‐corrected values for total binding at each time point. Sixty minutes after addition of 200 nM PSB603‐BY630, 10 μM MRS‐1706 was added to initial dissociation of the fluorescent ligand. Values show mean ± S.E.M of quadruplicate determinations in a single representative experiment. Similar data were obtained in four other experiments. The data points for the dissociation phase of the experiment were then fitted to a single exponential function to determine the the dissociation rate constant ( k off ) in min −1 as described under Methods. In this representative experiment the calculated K off value was 0.056 min −1 . The mean K off value obtained in the five repeat experiments was 0.065 ± 0.003 min ‐1 .

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: Ligand‐binding kinetics of 200 nM PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. BRET ratios for the total binding of 200 nM PSB603‐BY630 were obtained every 30 sec. In parallel, data were also collected in the absence of fluorescent ligand for each time point and these data were subtracted from the total binding to obtain baseline‐corrected values for total binding at each time point. Sixty minutes after addition of 200 nM PSB603‐BY630, 10 μM MRS‐1706 was added to initial dissociation of the fluorescent ligand. Values show mean ± S.E.M of quadruplicate determinations in a single representative experiment. Similar data were obtained in four other experiments. The data points for the dissociation phase of the experiment were then fitted to a single exponential function to determine the the dissociation rate constant ( k off ) in min −1 as described under Methods. In this representative experiment the calculated K off value was 0.056 min −1 . The mean K off value obtained in the five repeat experiments was 0.065 ± 0.003 min ‐1 .

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Ligand Binding Assay, Expressing, Binding Assay

    NanoBRET binding curves for PSB603‐BY630 and receptor‐selective fluorescent ligands binding to NLuc‐tagged A 1 , A 2A or A 3 adenosine receptors. (A, B) Total and non‐specific binding of (A) PSB603‐BY630 or (B) EC‐005 to transiently transfected NLuc‐A 2A R obtained in the absence and presence of 1 μM of the A 2A R‐selective antagonist SCH58261. Data are mean ± S.E.M obtained in five independent experiments (each conducted in duplicate). (C, D) Total and non‐specific binding of (C) PSB603‐BY630 or (D) EC‐069 to NLuc‐A 1 R in a stable HEK293T cell line obtained in the absence and presence of 1 μM of the A 1 R‐selective antagonist SLV320. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). (E, F) Total and non‐specific binding of (E) PSB603‐BY630 or (F) AV‐039 to NLuc‐A 3 R in a stable HEK293G cell line obtained in the absence and presence of 1 μM of the A 3 R‐selective antagonist MRS‐1220. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate).

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: NanoBRET binding curves for PSB603‐BY630 and receptor‐selective fluorescent ligands binding to NLuc‐tagged A 1 , A 2A or A 3 adenosine receptors. (A, B) Total and non‐specific binding of (A) PSB603‐BY630 or (B) EC‐005 to transiently transfected NLuc‐A 2A R obtained in the absence and presence of 1 μM of the A 2A R‐selective antagonist SCH58261. Data are mean ± S.E.M obtained in five independent experiments (each conducted in duplicate). (C, D) Total and non‐specific binding of (C) PSB603‐BY630 or (D) EC‐069 to NLuc‐A 1 R in a stable HEK293T cell line obtained in the absence and presence of 1 μM of the A 1 R‐selective antagonist SLV320. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). (E, F) Total and non‐specific binding of (E) PSB603‐BY630 or (F) AV‐039 to NLuc‐A 3 R in a stable HEK293G cell line obtained in the absence and presence of 1 μM of the A 3 R‐selective antagonist MRS‐1220. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate).

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Binding Assay, Transfection

    Effect of PSB603‐BY630 on Glosensor cAMP concentration‐response curves to the A 2B ‐selective agonist BAY 60–6583 in (A) HEK293G cells endogenously expressing A 2B R or (B) HEK293G cells overexpressing NLuc‐A 2B R. Concentration response curves were obtained in the absence and presence of 20 nM or 200 nM PSB603‐BY630. Values are mean ± S.E.M. of five separate experiments carried out in triplicate. Data represent peak luminescence response and are expressed as a percentage of the peak luminescence response to 3 μM BAY 60–6583 (in a) or 0.3 μM BAY 60–6583 (in b) obtained in the absence of antagonist in each individual experiment.

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: Effect of PSB603‐BY630 on Glosensor cAMP concentration‐response curves to the A 2B ‐selective agonist BAY 60–6583 in (A) HEK293G cells endogenously expressing A 2B R or (B) HEK293G cells overexpressing NLuc‐A 2B R. Concentration response curves were obtained in the absence and presence of 20 nM or 200 nM PSB603‐BY630. Values are mean ± S.E.M. of five separate experiments carried out in triplicate. Data represent peak luminescence response and are expressed as a percentage of the peak luminescence response to 3 μM BAY 60–6583 (in a) or 0.3 μM BAY 60–6583 (in b) obtained in the absence of antagonist in each individual experiment.

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Concentration Assay, Expressing

    Log EC 50 and E MAX values obtained in  HEK293G  cells endogenously expressing A 2B R or  HEK293G  cells expressing recombinant human A 2B R for BAY 60–6593 obtained in the absence and presence of increasing concentrations of PSB603BY630.

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: Log EC 50 and E MAX values obtained in HEK293G cells endogenously expressing A 2B R or HEK293G cells expressing recombinant human A 2B R for BAY 60–6593 obtained in the absence and presence of increasing concentrations of PSB603BY630.

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Expressing, Recombinant

    NanoBRET binding curves for PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: NanoBRET binding curves for PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Binding Assay, Expressing, Incubation

    NanoBRET imaging of PSB603‐BY630 binding to HEK293G cells expressing NLuc‐A 2B R. Cells were incubated with 100 nM PSB603‐BY630 in the absence (A, B) or presence (C, D) of 10 μM MRS‐1706 before addition of furimazine (1:800 dilution) and imaging. Filtered bioluminescence was captured using a 438/24 bandpass filter (cyan A & C). BRET was captured using a 650/50 nm bandpass filter (magenta B & D). Images are representative of those obtained in five independent experiments. Scale bar represents 100 μm.

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: NanoBRET imaging of PSB603‐BY630 binding to HEK293G cells expressing NLuc‐A 2B R. Cells were incubated with 100 nM PSB603‐BY630 in the absence (A, B) or presence (C, D) of 10 μM MRS‐1706 before addition of furimazine (1:800 dilution) and imaging. Filtered bioluminescence was captured using a 438/24 bandpass filter (cyan A & C). BRET was captured using a 650/50 nm bandpass filter (magenta B & D). Images are representative of those obtained in five independent experiments. Scale bar represents 100 μm.

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Imaging, Binding Assay, Expressing, Incubation

    NanoBRET competition binding in HEK293G cells exogenously expressing NLuc‐A 2B R. Cells were incubated with 50 nM PSB603‐BY630 in the absence or presence of competing ligands Data are mean ± S.E.M. from five independent experiments. The open and closed bars show the BRET ratio obtained in the absence and presence of 50 nM PSB603‐BY630 respectively.

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: NanoBRET competition binding in HEK293G cells exogenously expressing NLuc‐A 2B R. Cells were incubated with 50 nM PSB603‐BY630 in the absence or presence of competing ligands Data are mean ± S.E.M. from five independent experiments. The open and closed bars show the BRET ratio obtained in the absence and presence of 50 nM PSB603‐BY630 respectively.

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Binding Assay, Expressing, Incubation

    Ligand‐binding kinetics of 200 nM PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. BRET ratios for the total binding of 200 nM PSB603‐BY630 were obtained every 30 sec. In parallel, data were also collected in the absence of fluorescent ligand for each time point and these data were subtracted from the total binding to obtain baseline‐corrected values for total binding at each time point. Sixty minutes after addition of 200 nM PSB603‐BY630, 10 μM MRS‐1706 was added to initial dissociation of the fluorescent ligand. Values show mean ± S.E.M of quadruplicate determinations in a single representative experiment. Similar data were obtained in four other experiments. The data points for the dissociation phase of the experiment were then fitted to a single exponential function to determine the the dissociation rate constant ( k off ) in min −1 as described under Methods. In this representative experiment the calculated K off value was 0.056 min −1 . The mean K off value obtained in the five repeat experiments was 0.065 ± 0.003 min ‐1 .

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: Ligand‐binding kinetics of 200 nM PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. BRET ratios for the total binding of 200 nM PSB603‐BY630 were obtained every 30 sec. In parallel, data were also collected in the absence of fluorescent ligand for each time point and these data were subtracted from the total binding to obtain baseline‐corrected values for total binding at each time point. Sixty minutes after addition of 200 nM PSB603‐BY630, 10 μM MRS‐1706 was added to initial dissociation of the fluorescent ligand. Values show mean ± S.E.M of quadruplicate determinations in a single representative experiment. Similar data were obtained in four other experiments. The data points for the dissociation phase of the experiment were then fitted to a single exponential function to determine the the dissociation rate constant ( k off ) in min −1 as described under Methods. In this representative experiment the calculated K off value was 0.056 min −1 . The mean K off value obtained in the five repeat experiments was 0.065 ± 0.003 min ‐1 .

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Ligand Binding Assay, Expressing, Binding Assay

    NanoBRET binding curves for PSB603‐BY630 and receptor‐selective fluorescent ligands binding to NLuc‐tagged A 1 , A 2A or A 3 adenosine receptors. (A, B) Total and non‐specific binding of (A) PSB603‐BY630 or (B) EC‐005 to transiently transfected NLuc‐A 2A R obtained in the absence and presence of 1 μM of the A 2A R‐selective antagonist SCH58261. Data are mean ± S.E.M obtained in five independent experiments (each conducted in duplicate). (C, D) Total and non‐specific binding of (C) PSB603‐BY630 or (D) EC‐069 to NLuc‐A 1 R in a stable HEK293T cell line obtained in the absence and presence of 1 μM of the A 1 R‐selective antagonist SLV320. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). (E, F) Total and non‐specific binding of (E) PSB603‐BY630 or (F) AV‐039 to NLuc‐A 3 R in a stable HEK293G cell line obtained in the absence and presence of 1 μM of the A 3 R‐selective antagonist MRS‐1220. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate).

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: NanoBRET binding curves for PSB603‐BY630 and receptor‐selective fluorescent ligands binding to NLuc‐tagged A 1 , A 2A or A 3 adenosine receptors. (A, B) Total and non‐specific binding of (A) PSB603‐BY630 or (B) EC‐005 to transiently transfected NLuc‐A 2A R obtained in the absence and presence of 1 μM of the A 2A R‐selective antagonist SCH58261. Data are mean ± S.E.M obtained in five independent experiments (each conducted in duplicate). (C, D) Total and non‐specific binding of (C) PSB603‐BY630 or (D) EC‐069 to NLuc‐A 1 R in a stable HEK293T cell line obtained in the absence and presence of 1 μM of the A 1 R‐selective antagonist SLV320. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). (E, F) Total and non‐specific binding of (E) PSB603‐BY630 or (F) AV‐039 to NLuc‐A 3 R in a stable HEK293G cell line obtained in the absence and presence of 1 μM of the A 3 R‐selective antagonist MRS‐1220. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate).

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Binding Assay, Transfection

    Effect of PSB603‐BY630 on Glosensor cAMP concentration‐response curves to the A 2B ‐selective agonist BAY 60–6583 in (A) HEK293G cells endogenously expressing A 2B R or (B) HEK293G cells overexpressing NLuc‐A 2B R. Concentration response curves were obtained in the absence and presence of 20 nM or 200 nM PSB603‐BY630. Values are mean ± S.E.M. of five separate experiments carried out in triplicate. Data represent peak luminescence response and are expressed as a percentage of the peak luminescence response to 3 μM BAY 60–6583 (in a) or 0.3 μM BAY 60–6583 (in b) obtained in the absence of antagonist in each individual experiment.

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: Effect of PSB603‐BY630 on Glosensor cAMP concentration‐response curves to the A 2B ‐selective agonist BAY 60–6583 in (A) HEK293G cells endogenously expressing A 2B R or (B) HEK293G cells overexpressing NLuc‐A 2B R. Concentration response curves were obtained in the absence and presence of 20 nM or 200 nM PSB603‐BY630. Values are mean ± S.E.M. of five separate experiments carried out in triplicate. Data represent peak luminescence response and are expressed as a percentage of the peak luminescence response to 3 μM BAY 60–6583 (in a) or 0.3 μM BAY 60–6583 (in b) obtained in the absence of antagonist in each individual experiment.

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Concentration Assay, Expressing

    Log EC 50 and E MAX values obtained in  HEK293G  cells endogenously expressing A 2B R or  HEK293G  cells expressing recombinant human A 2B R for BAY 60–6593 obtained in the absence and presence of increasing concentrations of PSB603BY630.

    Journal: Pharmacology Research & Perspectives

    Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

    doi: 10.1002/prp2.1223

    Figure Lengend Snippet: Log EC 50 and E MAX values obtained in HEK293G cells endogenously expressing A 2B R or HEK293G cells expressing recombinant human A 2B R for BAY 60–6593 obtained in the absence and presence of increasing concentrations of PSB603BY630.

    Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

    Techniques: Expressing, Recombinant