Figure S3.
Characterization of GFP-EHD2-Cav1-mCh and Pac2-GFP-Cav1-mCh HeLa FlpIn cells and localization of Dyn2-GFP following EHD2 or Pac2 depletion. (A) Representative image from TIRF movie of a GFP-EHD2-Cav1-mCh cell. Red arrow depicts structures only positive for Cav1-mCh, green arrow highlights spots only positive for GFP-EHD2, and yellow arrows highlight structures positive for both GFP-EHD2 and Cav1-mCh. Immunoblot of GFP-EHD2-Cav1-mCh cells treated with concentrations of doxycycline as indicated. Doxycycline concentration used for further experiments were 1.0 ng/ml. (B) Representative image from TIRF movie of Pac2-GFP-Cav1-mCh cell. Red arrow depicts structures only positive for Cav1-mCh, green arrow highlights spots only positive for Pac2-GFP, and yellow arrows highlight structure positive for both Pac2-GFP and Cav1-mCh. Immunoblot of Pac2-GFP-Cav1-mCh cells treated with concentrations of doxycycline as indicated. Doxycycline concentration used for further experiments was 1.0 ng/ml. (C) Immunoblots of cells treated with siRNAs as indicated. GAPDH served as a loading control. (D) Representative image from TIRF movie of Dyn2-GFP-Cav1-mCh cells treated with siRNA directed against EHD2 or Pac2 as indicated. Yellow arrows highlight caveolae positive for Dyn2-GFP. (E) Quantification of Cav1-mCh track mean speed 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. Significance was assessed using t test, ***P ≤ 0.001. All scale bars, 10 μm. (F) FLIM-FRET of GFP-fusion proteins. Top images show fluorescence micrographs of cells expressing GFP-fusion proteins and Cav1-mCh as indicated. Bottom of the two panels show EGFP fluorescence lifetime. White arrows highlight structures where EGFP and mCherry colocalize. Scale bar, 2 μm. Source data are available for this figure: SourceData FS3.

Characterization of GFP-EHD2-Cav1-mCh and Pac2-GFP-Cav1-mCh HeLa FlpIn cells and localization of Dyn2-GFP following EHD2 or Pac2 depletion. (A) Representative image from TIRF movie of a GFP-EHD2-Cav1-mCh cell. Red arrow depicts structures only positive for Cav1-mCh, green arrow highlights spots only positive for GFP-EHD2, and yellow arrows highlight structures positive for both GFP-EHD2 and Cav1-mCh. Immunoblot of GFP-EHD2-Cav1-mCh cells treated with concentrations of doxycycline as indicated. Doxycycline concentration used for further experiments were 1.0 ng/ml. (B) Representative image from TIRF movie of Pac2-GFP-Cav1-mCh cell. Red arrow depicts structures only positive for Cav1-mCh, green arrow highlights spots only positive for Pac2-GFP, and yellow arrows highlight structure positive for both Pac2-GFP and Cav1-mCh. Immunoblot of Pac2-GFP-Cav1-mCh cells treated with concentrations of doxycycline as indicated. Doxycycline concentration used for further experiments was 1.0 ng/ml. (C) Immunoblots of cells treated with siRNAs as indicated. GAPDH served as a loading control. (D) Representative image from TIRF movie of Dyn2-GFP-Cav1-mCh cells treated with siRNA directed against EHD2 or Pac2 as indicated. Yellow arrows highlight caveolae positive for Dyn2-GFP. (E) Quantification of Cav1-mCh track mean speed 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. Significance was assessed using t test, ***P ≤ 0.001. All scale bars, 10 μm. (F) FLIM-FRET of GFP-fusion proteins. Top images show fluorescence micrographs of cells expressing GFP-fusion proteins and Cav1-mCh as indicated. Bottom of the two panels show EGFP fluorescence lifetime. White arrows highlight structures where EGFP and mCherry colocalize. Scale bar, 2 μm. Source data are available for this figure: SourceData FS3.

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