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The optimized dimericTomato-2xrGBD Rho sensor has the best location efficiency. (A) Spinning disk still images of a HeLa cells expressing the CMVdel-mNeonGreen-1xrGBD Rho sensor (upper panel) or CMVdel-dimericTomato-2xrGBD Rho sensor (lower panel) and H1R (not shown), which were stimulated with 100 μM histamine after 150 s and 10 μM pyrilamine after 350 s. The images match with the perturbation that is indicated for the plot in C. Times are min:s from the start of the recording. Scale bars: 25 µm. (B) Change in cytosolic intensity for CMV-mNeonGreen, CMV-eGFP-1xrGBD, CMVdel-3xmNeonGreen-1xrGBD, CMVdel-mNeonGreen-1xrGBD, -2xrGBD and <t>-3xrGBD,</t> and CMVdel-dimericTomato-1xrGBD and -2xrGBD (schematics on left) in HeLa cells expressing H1R, upon stimulation with 100 μM histamine. Each dot represents an individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The gray dashed line indicates no change in cytosolic intensity. The data is from two biological replicates based on two independent transfections. The number of cells per condition is: 3xmNG-1xrGBD=27, dT-1xrGBD=32, dT-2xrGBD=33, eGFP-1xrGBD=40, mNG=39, mNG-1xrGBD=28, mNG-2xrGBD=34, mNG-3xrGBD=26. (C) Time traces of the change in cytosolic intensity for the displayed cells for the mNeonGreen-1xrGBD sensor in gray and for the dimericTomato-2xrGBD sensor in black. mNG, mNeonGreen; dT, dimericTomato; rGBD, rhotekin G protein binding domain.
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The optimized dimericTomato-2xrGBD Rho sensor has the best location efficiency. (A) Spinning disk still images of a HeLa cells expressing the CMVdel-mNeonGreen-1xrGBD Rho sensor (upper panel) or CMVdel-dimericTomato-2xrGBD Rho sensor (lower panel) and H1R (not shown), which were stimulated with 100 μM histamine after 150 s and 10 μM pyrilamine after 350 s. The images match with the perturbation that is indicated for the plot in C. Times are min:s from the start of the recording. Scale bars: 25 µm. (B) Change in cytosolic intensity for CMV-mNeonGreen, CMV-eGFP-1xrGBD, CMVdel-3xmNeonGreen-1xrGBD, CMVdel-mNeonGreen-1xrGBD, -2xrGBD and -3xrGBD, and CMVdel-dimericTomato-1xrGBD and -2xrGBD (schematics on left) in HeLa cells expressing H1R, upon stimulation with 100 μM histamine. Each dot represents an individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The gray dashed line indicates no change in cytosolic intensity. The data is from two biological replicates based on two independent transfections. The number of cells per condition is: 3xmNG-1xrGBD=27, dT-1xrGBD=32, dT-2xrGBD=33, eGFP-1xrGBD=40, mNG=39, mNG-1xrGBD=28, mNG-2xrGBD=34, mNG-3xrGBD=26. (C) Time traces of the change in cytosolic intensity for the displayed cells for the mNeonGreen-1xrGBD sensor in gray and for the dimericTomato-2xrGBD sensor in black. mNG, mNeonGreen; dT, dimericTomato; rGBD, rhotekin G protein binding domain.

Journal: Journal of Cell Science

Article Title: Visualizing endogenous Rho activity with an improved localization-based, genetically encoded biosensor

doi: 10.1242/jcs.258823

Figure Lengend Snippet: The optimized dimericTomato-2xrGBD Rho sensor has the best location efficiency. (A) Spinning disk still images of a HeLa cells expressing the CMVdel-mNeonGreen-1xrGBD Rho sensor (upper panel) or CMVdel-dimericTomato-2xrGBD Rho sensor (lower panel) and H1R (not shown), which were stimulated with 100 μM histamine after 150 s and 10 μM pyrilamine after 350 s. The images match with the perturbation that is indicated for the plot in C. Times are min:s from the start of the recording. Scale bars: 25 µm. (B) Change in cytosolic intensity for CMV-mNeonGreen, CMV-eGFP-1xrGBD, CMVdel-3xmNeonGreen-1xrGBD, CMVdel-mNeonGreen-1xrGBD, -2xrGBD and -3xrGBD, and CMVdel-dimericTomato-1xrGBD and -2xrGBD (schematics on left) in HeLa cells expressing H1R, upon stimulation with 100 μM histamine. Each dot represents an individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The gray dashed line indicates no change in cytosolic intensity. The data is from two biological replicates based on two independent transfections. The number of cells per condition is: 3xmNG-1xrGBD=27, dT-1xrGBD=32, dT-2xrGBD=33, eGFP-1xrGBD=40, mNG=39, mNG-1xrGBD=28, mNG-2xrGBD=34, mNG-3xrGBD=26. (C) Time traces of the change in cytosolic intensity for the displayed cells for the mNeonGreen-1xrGBD sensor in gray and for the dimericTomato-2xrGBD sensor in black. mNG, mNeonGreen; dT, dimericTomato; rGBD, rhotekin G protein binding domain.

Article Snippet: The following plasmids, generated in this study, are available on Addgene ( http://www.addgene.org/ ): 129625 , dTomato-2xrGBD (C1 vector); 176098 , dimericTomato-2xrGBD (pLV vector); 129624 , mNeongreen-2xrGBD; 176091 , mNeonGreen-3xrGBD; 129633 , mNeonGreen-aGBD (anillin); 129634 , mNeonGreen-pGBD (PKN1 codon optimized); 176094 , H2A-mTurquoise2-CDC42-G12V-ΔCaaX; 176095 , H2A-mTurquoise2-RAC1-G12V-ΔCaaX; and 176097 , H2A-mTurquoise2-RHOA-G14V-ΔCaaX.

Techniques: Expressing, Transfection, Protein Binding