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  • 99
    New England Biolabs alwni
    Supplemental termination data for p[empty] experiments (A) Cartoon depicting the XmnI and <t>AlwNI</t> sites on p[ empty ], which are used for the dissolution and ligation assays, respectively, and the FLK2 locus, which is used for ChIP. (B) Plasmid <t>DNA</t> without a lacO array (p[ empty ]) was replicated and at different times chromatin was subjected to MCM7 and CDC45 ChIP. Percent recovery of FLK2 was quantified and used to measure dissociation of MCM7 and CDC45 (see methods). Dissolution and ligation were also quantified in parallel. mean±s.d. is plotted (n=3). The MCM7 and CDC45 dissociation data is obtained from the vehicle controls in Figure 5B–C , while the dissolution and ligation data are obtained from the vehicle controls in Fig 5D–E . (C) To seek independent evidence for the conclusions of the ChIP data presented in Figure 5B–C , we used a plasmid pull-down procedure. p[empty] was replicated in egg extracts treated with Vehicle or Ub-VS. At the indicated times, chromatin-associated proteins were captured on LacR-coated beads (which binds DNA independently of lacO sites) and analyzed by Western blotting for CDC45, MCM7, and PCNA. CDC45 and MCM7 dissociated from chromatin by 8’ in the vehicle control, but persisted following UbVS treatment. (D) To test whether the MCM7 modifications detected in panel (C) represented ubiquitylation, extracts were incubated with HIS 6 -Ubiquitin in the absence of Cyclin A, and in the absence or presence of plasmid DNA. After 15 minutes, HIS 6 -tagged proteins were captured by nickel resin pull down and blotted for MCM7. DNA replication greatly increased the levels of ubiquitylated MCM7, with the exception of a single species that was ubiquitylated independently of DNA replication (*). These data show that MCM7 is ubiquitylated during plasmid replication in egg extracts, as observed in yeast and during replication of sperm chromatin following nuclear assembly in egg extracts 24 , 25 . (E) In parallel to the plasmid pull-downs performed in (C), DNA samples were withdrawn for dissolution, ligation, and decatenation assays, none of which were perturbed by UbVS treatment. These data support our conclusion, based on ChIP experiments ( Fig. 5 ), that defective CMG unloading does not affect dissolution, ligation, or decatenation. (F) Decatenation was measured in the same reactions used to measure Dissolution and Ligation ( Fig. 5D–E ), mean±s.d. is plotted (n=3). (G–I) Given the experimental variability at the 4 minute time point in Figures 5D–F , the primary data and quantification for dissolution (G), ligation (H), and decatenation (I) for one of the three experiments summarized in Figure 5D–F is presented. This reveals that Ub-VS does not inhibit dissolution, ligation, or decatenation at the 4 minute time point. The same conclusion applies to two additional repetitions of this experiment (data not shown). (J) The primary ChIP data used to measure dissociation of MCM7 and CDC45 in Fig 5B–C is shown. Recovery of FLK2 was measured. mean±s.d. is plotted (n=3).
    Alwni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher caii caii
    MCT1 and <t>CAII</t> are co-localized in MCF-7 breast cancer cells. ( A ) Antibody staining of CAII ( A 1 ) and MCT1 ( A 2 ) in MCF-7 cells. ( A 3 ) Overlay of the fluorescence signals for MCT1 (red), CAII (green) and the nuclei marker DAPI (blue). The specificity of the primary antibodies was tested by incubating MCF-7 cells only with secondary antibodies ( A 4 ). ( B ) In situ proximity ligation assay (PLA) of MCT1 and CAII in MCF-7 breast cancer cells, incubated under normoxia ( B 1 ) and hypoxia ( B 2 ), respectively, and normoxic MCF-7 cells in which CAII was knocked down using <t>siRNA</t> ( B 3 ). The red dots indicate co-localization of MCT1 and CAII with a maximum distance of
    Caii Caii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher alwni enzyme
    MCT1 and <t>CAII</t> are co-localized in MCF-7 breast cancer cells. ( A ) Antibody staining of CAII ( A 1 ) and MCT1 ( A 2 ) in MCF-7 cells. ( A 3 ) Overlay of the fluorescence signals for MCT1 (red), CAII (green) and the nuclei marker DAPI (blue). The specificity of the primary antibodies was tested by incubating MCF-7 cells only with secondary antibodies ( A 4 ). ( B ) In situ proximity ligation assay (PLA) of MCT1 and CAII in MCF-7 breast cancer cells, incubated under normoxia ( B 1 ) and hypoxia ( B 2 ), respectively, and normoxic MCF-7 cells in which CAII was knocked down using <t>siRNA</t> ( B 3 ). The red dots indicate co-localization of MCT1 and CAII with a maximum distance of
    Alwni Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher alwni restriction endonuclease
    MCT1 and <t>CAII</t> are co-localized in MCF-7 breast cancer cells. ( A ) Antibody staining of CAII ( A 1 ) and MCT1 ( A 2 ) in MCF-7 cells. ( A 3 ) Overlay of the fluorescence signals for MCT1 (red), CAII (green) and the nuclei marker DAPI (blue). The specificity of the primary antibodies was tested by incubating MCF-7 cells only with secondary antibodies ( A 4 ). ( B ) In situ proximity ligation assay (PLA) of MCT1 and CAII in MCF-7 breast cancer cells, incubated under normoxia ( B 1 ) and hypoxia ( B 2 ), respectively, and normoxic MCF-7 cells in which CAII was knocked down using <t>siRNA</t> ( B 3 ). The red dots indicate co-localization of MCT1 and CAII with a maximum distance of
    Alwni Restriction Endonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher alwni digested pcrii plasmid
    MCT1 and <t>CAII</t> are co-localized in MCF-7 breast cancer cells. ( A ) Antibody staining of CAII ( A 1 ) and MCT1 ( A 2 ) in MCF-7 cells. ( A 3 ) Overlay of the fluorescence signals for MCT1 (red), CAII (green) and the nuclei marker DAPI (blue). The specificity of the primary antibodies was tested by incubating MCF-7 cells only with secondary antibodies ( A 4 ). ( B ) In situ proximity ligation assay (PLA) of MCT1 and CAII in MCF-7 breast cancer cells, incubated under normoxia ( B 1 ) and hypoxia ( B 2 ), respectively, and normoxic MCF-7 cells in which CAII was knocked down using <t>siRNA</t> ( B 3 ). The red dots indicate co-localization of MCT1 and CAII with a maximum distance of
    Alwni Digested Pcrii Plasmid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher fastdigest alwni caii
    MCT1 and <t>CAII</t> are co-localized in MCF-7 breast cancer cells. ( A ) Antibody staining of CAII ( A 1 ) and MCT1 ( A 2 ) in MCF-7 cells. ( A 3 ) Overlay of the fluorescence signals for MCT1 (red), CAII (green) and the nuclei marker DAPI (blue). The specificity of the primary antibodies was tested by incubating MCF-7 cells only with secondary antibodies ( A 4 ). ( B ) In situ proximity ligation assay (PLA) of MCT1 and CAII in MCF-7 breast cancer cells, incubated under normoxia ( B 1 ) and hypoxia ( B 2 ), respectively, and normoxic MCF-7 cells in which CAII was knocked down using <t>siRNA</t> ( B 3 ). The red dots indicate co-localization of MCT1 and CAII with a maximum distance of
    Fastdigest Alwni Caii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore alwni xhoi digested pet 31b
    MCT1 and <t>CAII</t> are co-localized in MCF-7 breast cancer cells. ( A ) Antibody staining of CAII ( A 1 ) and MCT1 ( A 2 ) in MCF-7 cells. ( A 3 ) Overlay of the fluorescence signals for MCT1 (red), CAII (green) and the nuclei marker DAPI (blue). The specificity of the primary antibodies was tested by incubating MCF-7 cells only with secondary antibodies ( A 4 ). ( B ) In situ proximity ligation assay (PLA) of MCT1 and CAII in MCF-7 breast cancer cells, incubated under normoxia ( B 1 ) and hypoxia ( B 2 ), respectively, and normoxic MCF-7 cells in which CAII was knocked down using <t>siRNA</t> ( B 3 ). The red dots indicate co-localization of MCT1 and CAII with a maximum distance of
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    Image Search Results


    Supplemental termination data for p[empty] experiments (A) Cartoon depicting the XmnI and AlwNI sites on p[ empty ], which are used for the dissolution and ligation assays, respectively, and the FLK2 locus, which is used for ChIP. (B) Plasmid DNA without a lacO array (p[ empty ]) was replicated and at different times chromatin was subjected to MCM7 and CDC45 ChIP. Percent recovery of FLK2 was quantified and used to measure dissociation of MCM7 and CDC45 (see methods). Dissolution and ligation were also quantified in parallel. mean±s.d. is plotted (n=3). The MCM7 and CDC45 dissociation data is obtained from the vehicle controls in Figure 5B–C , while the dissolution and ligation data are obtained from the vehicle controls in Fig 5D–E . (C) To seek independent evidence for the conclusions of the ChIP data presented in Figure 5B–C , we used a plasmid pull-down procedure. p[empty] was replicated in egg extracts treated with Vehicle or Ub-VS. At the indicated times, chromatin-associated proteins were captured on LacR-coated beads (which binds DNA independently of lacO sites) and analyzed by Western blotting for CDC45, MCM7, and PCNA. CDC45 and MCM7 dissociated from chromatin by 8’ in the vehicle control, but persisted following UbVS treatment. (D) To test whether the MCM7 modifications detected in panel (C) represented ubiquitylation, extracts were incubated with HIS 6 -Ubiquitin in the absence of Cyclin A, and in the absence or presence of plasmid DNA. After 15 minutes, HIS 6 -tagged proteins were captured by nickel resin pull down and blotted for MCM7. DNA replication greatly increased the levels of ubiquitylated MCM7, with the exception of a single species that was ubiquitylated independently of DNA replication (*). These data show that MCM7 is ubiquitylated during plasmid replication in egg extracts, as observed in yeast and during replication of sperm chromatin following nuclear assembly in egg extracts 24 , 25 . (E) In parallel to the plasmid pull-downs performed in (C), DNA samples were withdrawn for dissolution, ligation, and decatenation assays, none of which were perturbed by UbVS treatment. These data support our conclusion, based on ChIP experiments ( Fig. 5 ), that defective CMG unloading does not affect dissolution, ligation, or decatenation. (F) Decatenation was measured in the same reactions used to measure Dissolution and Ligation ( Fig. 5D–E ), mean±s.d. is plotted (n=3). (G–I) Given the experimental variability at the 4 minute time point in Figures 5D–F , the primary data and quantification for dissolution (G), ligation (H), and decatenation (I) for one of the three experiments summarized in Figure 5D–F is presented. This reveals that Ub-VS does not inhibit dissolution, ligation, or decatenation at the 4 minute time point. The same conclusion applies to two additional repetitions of this experiment (data not shown). (J) The primary ChIP data used to measure dissociation of MCM7 and CDC45 in Fig 5B–C is shown. Recovery of FLK2 was measured. mean±s.d. is plotted (n=3).

    Journal: Nature

    Article Title: The mechanism of DNA replication termination in vertebrates

    doi: 10.1038/nature14887

    Figure Lengend Snippet: Supplemental termination data for p[empty] experiments (A) Cartoon depicting the XmnI and AlwNI sites on p[ empty ], which are used for the dissolution and ligation assays, respectively, and the FLK2 locus, which is used for ChIP. (B) Plasmid DNA without a lacO array (p[ empty ]) was replicated and at different times chromatin was subjected to MCM7 and CDC45 ChIP. Percent recovery of FLK2 was quantified and used to measure dissociation of MCM7 and CDC45 (see methods). Dissolution and ligation were also quantified in parallel. mean±s.d. is plotted (n=3). The MCM7 and CDC45 dissociation data is obtained from the vehicle controls in Figure 5B–C , while the dissolution and ligation data are obtained from the vehicle controls in Fig 5D–E . (C) To seek independent evidence for the conclusions of the ChIP data presented in Figure 5B–C , we used a plasmid pull-down procedure. p[empty] was replicated in egg extracts treated with Vehicle or Ub-VS. At the indicated times, chromatin-associated proteins were captured on LacR-coated beads (which binds DNA independently of lacO sites) and analyzed by Western blotting for CDC45, MCM7, and PCNA. CDC45 and MCM7 dissociated from chromatin by 8’ in the vehicle control, but persisted following UbVS treatment. (D) To test whether the MCM7 modifications detected in panel (C) represented ubiquitylation, extracts were incubated with HIS 6 -Ubiquitin in the absence of Cyclin A, and in the absence or presence of plasmid DNA. After 15 minutes, HIS 6 -tagged proteins were captured by nickel resin pull down and blotted for MCM7. DNA replication greatly increased the levels of ubiquitylated MCM7, with the exception of a single species that was ubiquitylated independently of DNA replication (*). These data show that MCM7 is ubiquitylated during plasmid replication in egg extracts, as observed in yeast and during replication of sperm chromatin following nuclear assembly in egg extracts 24 , 25 . (E) In parallel to the plasmid pull-downs performed in (C), DNA samples were withdrawn for dissolution, ligation, and decatenation assays, none of which were perturbed by UbVS treatment. These data support our conclusion, based on ChIP experiments ( Fig. 5 ), that defective CMG unloading does not affect dissolution, ligation, or decatenation. (F) Decatenation was measured in the same reactions used to measure Dissolution and Ligation ( Fig. 5D–E ), mean±s.d. is plotted (n=3). (G–I) Given the experimental variability at the 4 minute time point in Figures 5D–F , the primary data and quantification for dissolution (G), ligation (H), and decatenation (I) for one of the three experiments summarized in Figure 5D–F is presented. This reveals that Ub-VS does not inhibit dissolution, ligation, or decatenation at the 4 minute time point. The same conclusion applies to two additional repetitions of this experiment (data not shown). (J) The primary ChIP data used to measure dissociation of MCM7 and CDC45 in Fig 5B–C is shown. Recovery of FLK2 was measured. mean±s.d. is plotted (n=3).

    Article Snippet: To monitor ligation, 0.25–1.0 ng/µl of purified DNA was incubated in CutSmart buffer with 0.2 units/µl of AlwNI (New England BioLabs) at 37°C for 1 hour.

    Techniques: Ligation, Chromatin Immunoprecipitation, Plasmid Preparation, Western Blot, Incubation

    MCT1 and CAII are co-localized in MCF-7 breast cancer cells. ( A ) Antibody staining of CAII ( A 1 ) and MCT1 ( A 2 ) in MCF-7 cells. ( A 3 ) Overlay of the fluorescence signals for MCT1 (red), CAII (green) and the nuclei marker DAPI (blue). The specificity of the primary antibodies was tested by incubating MCF-7 cells only with secondary antibodies ( A 4 ). ( B ) In situ proximity ligation assay (PLA) of MCT1 and CAII in MCF-7 breast cancer cells, incubated under normoxia ( B 1 ) and hypoxia ( B 2 ), respectively, and normoxic MCF-7 cells in which CAII was knocked down using siRNA ( B 3 ). The red dots indicate co-localization of MCT1 and CAII with a maximum distance of

    Journal: eLife

    Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells

    doi: 10.7554/eLife.35176

    Figure Lengend Snippet: MCT1 and CAII are co-localized in MCF-7 breast cancer cells. ( A ) Antibody staining of CAII ( A 1 ) and MCT1 ( A 2 ) in MCF-7 cells. ( A 3 ) Overlay of the fluorescence signals for MCT1 (red), CAII (green) and the nuclei marker DAPI (blue). The specificity of the primary antibodies was tested by incubating MCF-7 cells only with secondary antibodies ( A 4 ). ( B ) In situ proximity ligation assay (PLA) of MCT1 and CAII in MCF-7 breast cancer cells, incubated under normoxia ( B 1 ) and hypoxia ( B 2 ), respectively, and normoxic MCF-7 cells in which CAII was knocked down using siRNA ( B 3 ). The red dots indicate co-localization of MCT1 and CAII with a maximum distance of

    Article Snippet: siRNA-mediated knockdown of CAII CAII was knocked down in MCF-7 cells using siRNA (Ambion Silencer Select anti CA2 siRNA, s2249, Life Technologies).

    Techniques: Staining, Fluorescence, Marker, In Situ, Proximity Ligation Assay, Incubation

    Determination of CAII knockdown efficiency in MCF-7 cells. ( A ) ΔCt values for CAII minus RPL27 of normoxic and hypoxic MCF-7 cells, treated with siRNA against CAII (blue bars) or non-targeting negative control siRNA (gray bars) (mean +SEM). *p≤0.05; Student’s t-test. ( B ) Relative change in the RNA level of CAII as given by the 2 –ΔΔCt values for CAII in normoxic and hypoxic MCF-7 cells treated with siRNA against CAII compared to cells treated with non-targeting negative control siRNA (mean +SEM). ( C ) Representative western blot for CAII (upper panel) and β-actin (lower panel) from normoxic and hypoxic MCF-7 cells, treated with siRNA against CAII or non-targeting negative control siRNA. ( D ) Relative intensity of the fluorescent signal for CAII, normalized to the signal intensity of β-actin in the same probe (mean +SEM). The significance indicators above the bars for siCAII refer to the values of siNeg. ***p≤0.001; Student’s t-test.

    Journal: eLife

    Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells

    doi: 10.7554/eLife.35176

    Figure Lengend Snippet: Determination of CAII knockdown efficiency in MCF-7 cells. ( A ) ΔCt values for CAII minus RPL27 of normoxic and hypoxic MCF-7 cells, treated with siRNA against CAII (blue bars) or non-targeting negative control siRNA (gray bars) (mean +SEM). *p≤0.05; Student’s t-test. ( B ) Relative change in the RNA level of CAII as given by the 2 –ΔΔCt values for CAII in normoxic and hypoxic MCF-7 cells treated with siRNA against CAII compared to cells treated with non-targeting negative control siRNA (mean +SEM). ( C ) Representative western blot for CAII (upper panel) and β-actin (lower panel) from normoxic and hypoxic MCF-7 cells, treated with siRNA against CAII or non-targeting negative control siRNA. ( D ) Relative intensity of the fluorescent signal for CAII, normalized to the signal intensity of β-actin in the same probe (mean +SEM). The significance indicators above the bars for siCAII refer to the values of siNeg. ***p≤0.001; Student’s t-test.

    Article Snippet: siRNA-mediated knockdown of CAII CAII was knocked down in MCF-7 cells using siRNA (Ambion Silencer Select anti CA2 siRNA, s2249, Life Technologies).

    Techniques: Negative Control, Western Blot

    CAII supports proliferation of MCF-7 breast cancer cells. ( A, B ) Staining of nuclei with Hoechst 33342 (blue) in MCF-7 cells after 3 days in culture under normoxic ( A ) or hypoxic ( B ) conditions. Cells were untreated (control; A1, B1 ), mock-transfected with non-targeting negative control siRNA (siNeg; A2, B2 ), transfected with siRNA against CAII (siCAII; A3, B3 ), incubated with 30 µM of the CA inhibitor EZA ( A4, B4 ), or incubated with 300 nM of the MCT1 inhibitor AR-C155858 ( A5, B5 ). ( C, D ) Total number of nuclei/mm 2 in normoxic ( C ) and hypoxic ( D ) MCF-7 cell cultures, kept for 0–3 days under the conditions described for ( A ) and ( B ). For every data point, four dishes of cells were used and three pictures were taken from each dish at random locations, yielding 12 pictures/data points (n = 12/4). The blue asterisks indicate significance in differences between siCAII and siNeg, the orange asterisks between control and AR-C155858, and the yellow significance indicators between control and EZA. *p≤0.05, **p≤0.01, ***p≤0.001, n.s. no significance; Student’s t-test. 10.7554/eLife.35176.010 Original dataset for Figure 3 .

    Journal: eLife

    Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells

    doi: 10.7554/eLife.35176

    Figure Lengend Snippet: CAII supports proliferation of MCF-7 breast cancer cells. ( A, B ) Staining of nuclei with Hoechst 33342 (blue) in MCF-7 cells after 3 days in culture under normoxic ( A ) or hypoxic ( B ) conditions. Cells were untreated (control; A1, B1 ), mock-transfected with non-targeting negative control siRNA (siNeg; A2, B2 ), transfected with siRNA against CAII (siCAII; A3, B3 ), incubated with 30 µM of the CA inhibitor EZA ( A4, B4 ), or incubated with 300 nM of the MCT1 inhibitor AR-C155858 ( A5, B5 ). ( C, D ) Total number of nuclei/mm 2 in normoxic ( C ) and hypoxic ( D ) MCF-7 cell cultures, kept for 0–3 days under the conditions described for ( A ) and ( B ). For every data point, four dishes of cells were used and three pictures were taken from each dish at random locations, yielding 12 pictures/data points (n = 12/4). The blue asterisks indicate significance in differences between siCAII and siNeg, the orange asterisks between control and AR-C155858, and the yellow significance indicators between control and EZA. *p≤0.05, **p≤0.01, ***p≤0.001, n.s. no significance; Student’s t-test. 10.7554/eLife.35176.010 Original dataset for Figure 3 .

    Article Snippet: siRNA-mediated knockdown of CAII CAII was knocked down in MCF-7 cells using siRNA (Ambion Silencer Select anti CA2 siRNA, s2249, Life Technologies).

    Techniques: Staining, Transfection, Negative Control, Incubation

    Influence of pH i on lactate transport. ( A ) Initial intracellular pH (pH i ), as measured at the beginning of the experiment, of normoxic (left column) and hypoxic (right column) MCF-7 cells, treated with siRNA against CAII (blue) or non-targeting negative control siRNA (gray) (mean ± SEM). ***p≤0.001; Student’s t-test. ( B–E ) ΔpH i /Δt during application of 3 mM lactate to normoxic ( B, C ) and hypoxic ( D, E ) MCF-7 cells, treated with either negative control siRNA ( B, D ) or CAII-siRNA ( C, E ), as shown in Figure 1B , plotted against the cells initial pH i . Every dot represents one individual cell. The red lines represent linear regression fits.

    Journal: eLife

    Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells

    doi: 10.7554/eLife.35176

    Figure Lengend Snippet: Influence of pH i on lactate transport. ( A ) Initial intracellular pH (pH i ), as measured at the beginning of the experiment, of normoxic (left column) and hypoxic (right column) MCF-7 cells, treated with siRNA against CAII (blue) or non-targeting negative control siRNA (gray) (mean ± SEM). ***p≤0.001; Student’s t-test. ( B–E ) ΔpH i /Δt during application of 3 mM lactate to normoxic ( B, C ) and hypoxic ( D, E ) MCF-7 cells, treated with either negative control siRNA ( B, D ) or CAII-siRNA ( C, E ), as shown in Figure 1B , plotted against the cells initial pH i . Every dot represents one individual cell. The red lines represent linear regression fits.

    Article Snippet: siRNA-mediated knockdown of CAII CAII was knocked down in MCF-7 cells using siRNA (Ambion Silencer Select anti CA2 siRNA, s2249, Life Technologies).

    Techniques: Negative Control