Analysis of Tf -647 uptake and caveolae track duration time in Cav1-mCh FlpIn cells depleted of Dyn1 or Dyn2. (A) Analysis of mean squared displacement over time of nine stable Cav1-mCh–positive caveolae. (B) Representative images of fixed Cav1-mCh cells transfected with siRNA as indicated for 72 h before 10 min incubation with Alexa fluor 647 conjugated Tf (Tf-647; 5 μg/ml) at 37°C. Scale bar, 10 μm. (C) Quantification shows relative Tf-647 fluorescent intensity/microns2. At least 40 cells were quantified per condition, significance was assessed using t test, ***P ≤ 0.001. (D) Immunoblots of whole cell lysate of Cav1-mCh cells treated with siRNA as indicated for 72 h prior to harvest. As a positive control protein, lysate of rat brain was loaded. Two exposures of the blot probed with rabbit anit-Dyn1 are shown to illustrate the extreme difference of Dyn1 levels present in the different cell types. Molecular weights in kD. (E and F) Quantification of the Cav1-mCh track duration time in cells depleted of Dyn1 using three independent siRNAs as indicated (E) or cells depleted of both Dyn1 and Dyn2 using siRNA as indicated. GAPDH served as a loading control (F). Numbers were related to ctrl siRNA treated cells. Analyses were performed using Imaris software, and track mean ± SEM from at least 12 (E) or 10 (F) cells per condition are shown. Significance was assessed using t test, ***P ≤ 0.001. Source data are available for this figure: SourceData FS1.
