Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Single particle tracking of CFTR in the plasma membrane of COS7, BHK and MDCK cells. A, CFTR diffusion in COS7 cells (top, data as in Fig. 4A, shown for reference) labeled with anti-HA Fab fragments (as opposed to anti-HA antibody, second panel), in BHK cells (third panel), and in MDCK cells (fourth panel). B, Representative trajectories for CFTR diffusion in the plasma membrane of COS7 cells labeled with anti-HA Fab fragment and Cy3-conjugated secondary Fab fragment (left, Cy3-conjugated goat anti-mouse Fab fragment) and from cells labeled with anti-HA antibody, secondary biotinylated Fab fragment and streptavidin conjugated Qdots (right). Data obtained at 30 fps. C, Distributions of D and range (at 2 s) for diffusion of CFTR-3HA (top two panels) and CFTR-3HA-Δ26 (bottom two panels) at indicated acquisition frame rates. Data at 10 fps in panel A shown for reference.
• Supplemental Video 1 - Long-term diffusion of CFTR-3HA in the plasma membrane of MDCK cells. MDCK cells expressing CFTR-3HA were labeled by sequential incubation with anti-HA antibody, biotinylated secondary Fab fragment and streptavidin Qdots. Movie represents 5 min of data (at 0.9 fps) and played ~10-times accelerated. The area shown is ~30 × 30 μm.
• Supplemental Video 2 - Long-term diffusion of CFTR-3HA in the plasma membrane of COS7 cells. COS7 cells transiently transfected to express CFTR-3HA were labeled by sequential incubation with anti-HA antibody, biotinylated secondary Fab fragment and streptavidin Qdots. Movie represents 5 min of data (at 0.9 fps) and played ~10-times accelerated. The area shown is ~30 × 30 μm.
• Supplemental Video 3 - Single particle tracking of CFTR-3HA in the plasma membrane of COS7 cells. COS7 cells transiently transfected to express CFTR-3HA were labeled by sequential incubation with anti-HA antibody, biotinylated secondary Fab fragment and streptavidin Qdots. Movie represents 1 min of data (collected at 10 fps) and played ~2.5-times accelerated. The area shown is ~30 × 30 μm.
• Supplemental Video 4 - Single particle tracking of CFTR-3HA in the plasma membrane of human bronchial epithelial cells. HBE cells transiently transfected to express CFTR-3HA were labeled by sequential incubation with anti-HA antibody, biotinylated secondary Fab fragment and streptavidin Qdots. Movie represents 1 min of data (collected at 10 fps) and played ~2.5-times accelerated. The area shown is ~30 × 25 μm.
• Supplemental Video 5 - Long-term diffusion of CFTR-3HA-Δ26 in the plasma membrane of COS7 cells. COS7 cells expressing CFTR-3HA-Δ26 were labeled by sequential incubation with anti-HA antibody, biotinylated secondary Fab fragment and streptavidin Qdots. Movie represents 5 min of data (at 0.9 fps) and played ~10-times accelerated. The area shown is ~30 × 30 μm.
• Supplemental Video 6 - Single particle tracking of CFTR-3HA-Δ70 in the plasma membrane of COS7 cells. COS7 cells transiently transfected to express CFTR-3HA-Δ70 were labeled by sequential incubation with anti-HA antibody, biotinylated secondary Fab fragment and streptavidin Qdots. Movie represents 1 min of data (collected at 10 fps) and played ~2.5-times accelerated. The area shown is ~24 × 24 μm.
• Supplemental Video 7 - Diffusion of CFTR-3HA in the plasma membrane of BHK cells in the presence of GFP-CFTRct87. BHK cells stably expressing CFTR-3HA were transiently transfected with plasmid encoding GFP-CFTRct87. Data acquired for 1 min (at 10 fps) using filters for 655 nm Qdots. GFP fluorescence ‘bleed-through’ can be seen (left cells) to identify cells expressing GFP-CFTRct87, whereas non-transfected cells have near-zero background. The area imaged is ~45 × 20 μm and the movie is played ~2.5-times accelerated.