Figure 2. Loss of β3 results in... | Rockefeller University Press

Figure 2.

$Figure 2. Loss of β3 results in formation of unstable actin protrusions. (A) WT and β3−/− cells analyzed for total F-actin content by FACS analysis of phalloidin staining intensity. Data are presented as the percentage intensity of WT control cells ± SEM pooled from three independent experiments (indicated by error bars). (B) WT, β3−/−, or β3-GFP cell lysates from TX-100 insoluble fractions analyzed by Western blotting for β-actin or GAPDH as a loading control. Graphs are representative of four experiments. (C) Kymography analysis of protrusion rate and the percentage of cell area protruding/frame from time-lapse videos. Still images and example of kymographs obtained from the example white lines are shown on the left. Six lines were quantified from 12 cells over three independent experiments. Data are mean ± SEM (indicated by error bars). (D) WT and β3−/− cells transfected with GFP-actin and imaged by fluorescence time-lapse microscopy. Stills are shown from an example of each cell type, enlarged images of protruding regions of membrane over time (boxes) are shown on the right. (E) Quantification and example images of free barbed end formation in WT and β3−/− cells. Live cells were pulsed with Alexa Fluor 568–G-actin (red) for 1 min, fixed, and stained with phalloidin-488 (green). The intensity of G-actin incorporation was quantified from at least 100 cells from three independent experiments. Data are shown as the mean percentage intensity ± SEM compared with WT control (indicated by error bars). **, P < 0.01. Bars, 10 µm.$

Loss of β3 results in formation of unstable actin protrusions. (A) WT and β3−/− cells analyzed for total F-actin content by FACS analysis of phalloidin staining intensity. Data are presented as the percentage intensity of WT control cells ± SEM pooled from three independent experiments (indicated by error bars). (B) WT, β3−/−, or β3-GFP cell lysates from TX-100 insoluble fractions analyzed by Western blotting for β-actin or GAPDH as a loading control. Graphs are representative of four experiments. (C) Kymography analysis of protrusion rate and the percentage of cell area protruding/frame from time-lapse videos. Still images and example of kymographs obtained from the example white lines are shown on the left. Six lines were quantified from 12 cells over three independent experiments. Data are mean ± SEM (indicated by error bars). (D) WT and β3−/− cells transfected with GFP-actin and imaged by fluorescence time-lapse microscopy. Stills are shown from an example of each cell type, enlarged images of protruding regions of membrane over time (boxes) are shown on the right. (E) Quantification and example images of free barbed end formation in WT and β3−/− cells. Live cells were pulsed with Alexa Fluor 568–G-actin (red) for 1 min, fixed, and stained with phalloidin-488 (green). The intensity of G-actin incorporation was quantified from at least 100 cells from three independent experiments. Data are shown as the mean percentage intensity ± SEM compared with WT control (indicated by error bars). **, P < 0.01. Bars, 10 µm.

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