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(A) A schematic summarizing the lysate assay protocol. PLCγ1-mNG was generated in HEK293T cells, then flowed directly as dilute lysate over pLAT bilayers containing PIP 2 . Active PLCγ1-mNG hydrolyzed PIP 2 to DAG, which was detected using a labeled DAG sensor (PKCθ-C1b-SNAP-Alexa 647). Parts of the figure were drawn using images from Servier Medical Art Commons Attribution 3.0 Unported License ( http://smart.servier.com (accessed on 21 March 2022)). (B) An example TIRF image of the DAG sensor demonstrating the segmentation of 50 x 50 µm images for analysis. TIRF images in a time series were sectioned into 5 x 5 µm patches and analyzed as separate instances. (C) Images of a single 5 x 5 µm patch in the DAG sensor channel (Top) and PLCγ1-mNG channel (Bottom) through time, corresponding to the time axis for the curves below. (D) Curves obtained from processing the raw median fluorescence from membrane patches, representing the amount of DAG in the bilayer (red, left axis, foreground curves) and density of PLCγ1-mNG recruited (green, right axis, background curves) over 20 minutes. PLCγ1-mNG lysate (∼1:1000 dilution) with DAG sensor was injected at t = 0 min. To attain quantitative curves, raw fluorescence was first normalized to the time of injection. DAG was calibrated using the assumption that the average normalized maximum for multiple experiments must be 2% (given 2% PIP 2 bilayers). PLCγ1-mNG was calibrated by matching single-molecule counts (at high laser power) to bulk fluorescence (at low laser power). The two solid, darkly colored curves represent the median of all patches analyzed. Lightly colored curves in the background represent the results from each single patch (n = 100). Lipid composition (mol%): 94:4:2 DOPC:Ni-DGS:PIP 2 . LAT density: ∼1500 µm -2 .
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(A) A schematic summarizing the lysate assay protocol. PLCγ1-mNG was generated in HEK293T cells, then flowed directly as dilute lysate over pLAT bilayers containing PIP 2 . Active PLCγ1-mNG hydrolyzed PIP 2 to DAG, which was detected using a labeled DAG sensor (PKCθ-C1b-SNAP-Alexa 647). Parts of the figure were drawn using images from Servier Medical Art Commons Attribution 3.0 Unported License ( http://smart.servier.com (accessed on 21 March 2022)). (B) An example TIRF image of the DAG sensor demonstrating the segmentation of 50 x 50 µm images for analysis. TIRF images in a time series were sectioned into 5 x 5 µm patches and analyzed as separate instances. (C) Images of a single 5 x 5 µm patch in the DAG sensor channel (Top) and PLCγ1-mNG channel (Bottom) through time, corresponding to the time axis for the curves below. (D) Curves obtained from processing the raw median fluorescence from membrane patches, representing the amount of DAG in the bilayer (red, left axis, foreground curves) and density of PLCγ1-mNG recruited (green, right axis, background curves) over 20 minutes. PLCγ1-mNG lysate (∼1:1000 dilution) with DAG sensor was injected at t = 0 min. To attain quantitative curves, raw fluorescence was first normalized to the time of injection. DAG was calibrated using the assumption that the average normalized maximum for multiple experiments must be 2% (given 2% PIP 2 bilayers). PLCγ1-mNG was calibrated by matching single-molecule counts (at high laser power) to bulk fluorescence (at low laser power). The two solid, darkly colored curves represent the median of all patches analyzed. Lightly colored curves in the background represent the results from each single patch (n = 100). Lipid composition (mol%): 94:4:2 DOPC:Ni-DGS:PIP 2 . LAT density: ∼1500 µm -2 .
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(A) A schematic summarizing the lysate assay protocol. PLCγ1-mNG was generated in HEK293T cells, then flowed directly as dilute lysate over pLAT bilayers containing PIP 2 . Active PLCγ1-mNG hydrolyzed PIP 2 to DAG, which was detected using a labeled DAG sensor (PKCθ-C1b-SNAP-Alexa 647). Parts of the figure were drawn using images from Servier Medical Art Commons Attribution 3.0 Unported License ( http://smart.servier.com (accessed on 21 March 2022)). (B) An example TIRF image of the DAG sensor demonstrating the segmentation of 50 x 50 µm images for analysis. TIRF images in a time series were sectioned into 5 x 5 µm patches and analyzed as separate instances. (C) Images of a single 5 x 5 µm patch in the DAG sensor channel (Top) and PLCγ1-mNG channel (Bottom) through time, corresponding to the time axis for the curves below. (D) Curves obtained from processing the raw median fluorescence from membrane patches, representing the amount of DAG in the bilayer (red, left axis, foreground curves) and density of PLCγ1-mNG recruited (green, right axis, background curves) over 20 minutes. PLCγ1-mNG lysate (∼1:1000 dilution) with DAG sensor was injected at t = 0 min. To attain quantitative curves, raw fluorescence was first normalized to the time of injection. DAG was calibrated using the assumption that the average normalized maximum for multiple experiments must be 2% (given 2% PIP 2 bilayers). PLCγ1-mNG was calibrated by matching single-molecule counts (at high laser power) to bulk fluorescence (at low laser power). The two solid, darkly colored curves represent the median of all patches analyzed. Lightly colored curves in the background represent the results from each single patch (n = 100). Lipid composition (mol%): 94:4:2 DOPC:Ni-DGS:PIP 2 . LAT density: ∼1500 µm -2 .

Journal: bioRxiv

Article Title: Supported membrane assay probes PLCγ1 activity in LAT condensates

doi: 10.64898/2026.01.23.701188

Figure Lengend Snippet: (A) A schematic summarizing the lysate assay protocol. PLCγ1-mNG was generated in HEK293T cells, then flowed directly as dilute lysate over pLAT bilayers containing PIP 2 . Active PLCγ1-mNG hydrolyzed PIP 2 to DAG, which was detected using a labeled DAG sensor (PKCθ-C1b-SNAP-Alexa 647). Parts of the figure were drawn using images from Servier Medical Art Commons Attribution 3.0 Unported License ( http://smart.servier.com (accessed on 21 March 2022)). (B) An example TIRF image of the DAG sensor demonstrating the segmentation of 50 x 50 µm images for analysis. TIRF images in a time series were sectioned into 5 x 5 µm patches and analyzed as separate instances. (C) Images of a single 5 x 5 µm patch in the DAG sensor channel (Top) and PLCγ1-mNG channel (Bottom) through time, corresponding to the time axis for the curves below. (D) Curves obtained from processing the raw median fluorescence from membrane patches, representing the amount of DAG in the bilayer (red, left axis, foreground curves) and density of PLCγ1-mNG recruited (green, right axis, background curves) over 20 minutes. PLCγ1-mNG lysate (∼1:1000 dilution) with DAG sensor was injected at t = 0 min. To attain quantitative curves, raw fluorescence was first normalized to the time of injection. DAG was calibrated using the assumption that the average normalized maximum for multiple experiments must be 2% (given 2% PIP 2 bilayers). PLCγ1-mNG was calibrated by matching single-molecule counts (at high laser power) to bulk fluorescence (at low laser power). The two solid, darkly colored curves represent the median of all patches analyzed. Lightly colored curves in the background represent the results from each single patch (n = 100). Lipid composition (mol%): 94:4:2 DOPC:Ni-DGS:PIP 2 . LAT density: ∼1500 µm -2 .

Article Snippet: PKCθ-C1b-SNAP (DAG sensor) was labeled with SNAP-Surface Alexa 647 (SNAP-Alexa 647, S9136S, New England Biolabs) according to the manufacturer protocol.

Techniques: Generated, Labeling, Fluorescence, Membrane, Injection