Figure 3.
Dyn2 and EHD2 stabilize caveolae via different but cooperative mechanisms. (A) Image panel showing GFP recovery after photobleaching in Dyn2GFP-Cav1-mCh cells, GFP-EHD2-Cav1-mCh cells, or Pac2-GFP-Cav1-mCh cells. Graph shows percentage GFP-intensity recovery curves after photobleaching of mCh and GFP colocalizing structures at the PM. Arrowheads depict caveolae prior to and after photobleaching as indicated in the figure. Scale bar, 2 μm. (B) Recovery curves of Dyn2-GFP intensities after photobleaching of caveolae in Dyn2-GFP-Cav1-mCh cells treated with Ctrl, EHD2, or Pac2 siRNA as indicated. (C) Quantification of Cav1-mCh track duration time and displacement length in GFP-EHD2-Cav1-mCh cells (green). Numbers were related to Cav1-mCh cells (white). Track mean ± SEM from at least seven cells per condition are shown. (D) Representative image from TIRF movie of Dyn2-GFP-Cav1-mCh cells transiently expressing EHD2-BFP. White arrows highlight structures where all three tagged proteins are colocalized, and green arrows highlights structures only positive for Dyn2-GFP. Scale bar, 10 or 2 μm for magnification. (E) Immunogold labeling of EHD2 (large gold) and Dyn2 (small gold) on PM lawns prepared from 3T3-L1 cells. Arrowheads depict caveolae positive for both EHD2 and Dyn2. Scale bar, 100 nm. (F) Quantification of Cav1-mCh track duration time and displacement length in Dyn2-GFP-Cav1-mCh cells (black), transiently expressing EHD2-BFP (dark green), or depleted from EHD2 (purple). Numbers were related to Dyn2-GFP-Cav1-mCh cells (black). Track mean ± SEM from at least nine cells per condition are shown. Significance was assessed using t test, **P ≤ 0.01, ***P ≤ 0.001.

Dyn2 and EHD2 stabilize caveolae via different but cooperative mechanisms. (A) Image panel showing GFP recovery after photobleaching in Dyn2GFP-Cav1-mCh cells, GFP-EHD2-Cav1-mCh cells, or Pac2-GFP-Cav1-mCh cells. Graph shows percentage GFP-intensity recovery curves after photobleaching of mCh and GFP colocalizing structures at the PM. Arrowheads depict caveolae prior to and after photobleaching as indicated in the figure. Scale bar, 2 μm. (B) Recovery curves of Dyn2-GFP intensities after photobleaching of caveolae in Dyn2-GFP-Cav1-mCh cells treated with Ctrl, EHD2, or Pac2 siRNA as indicated. (C) Quantification of Cav1-mCh track duration time and displacement length in GFP-EHD2-Cav1-mCh cells (green). Numbers were related to Cav1-mCh cells (white). Track mean ± SEM from at least seven cells per condition are shown. (D) Representative image from TIRF movie of Dyn2-GFP-Cav1-mCh cells transiently expressing EHD2-BFP. White arrows highlight structures where all three tagged proteins are colocalized, and green arrows highlights structures only positive for Dyn2-GFP. Scale bar, 10 or 2 μm for magnification. (E) Immunogold labeling of EHD2 (large gold) and Dyn2 (small gold) on PM lawns prepared from 3T3-L1 cells. Arrowheads depict caveolae positive for both EHD2 and Dyn2. Scale bar, 100 nm. (F) Quantification of Cav1-mCh track duration time and displacement length in Dyn2-GFP-Cav1-mCh cells (black), transiently expressing EHD2-BFP (dark green), or depleted from EHD2 (purple). Numbers were related to Dyn2-GFP-Cav1-mCh cells (black). Track mean ± SEM from at least nine cells per condition are shown. Significance was assessed using t test, **P ≤ 0.01, ***P ≤ 0.001.

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