JCS03336 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Overexpression levels of R1-EGFP variants. HeLa cells were transfected with various R1-EGFP constructs and cell lysates were analyzed by western blot with a reggie-1-specific antibody that detects both the endogenous (lower band) and transfected (upper band) protein. Quantification of the bands showed that R1-EGFP variants were overexpressed 0.62- to 1.3-fold.

• Movie 1 -

  Movie 1. R1-EGFP is endocytosed upon stimulation with EGF. HeLa cells transfected with R1-EGFP were stimulated with EGF and live imaging of the cells was performed with an interval of one frame per second with total of 300 frames. Pinhole settings correspond to an optical thickness z=1.5 μm. The play rate of the movie is 5 frames/second. A translocation of R1-EGFP into endosomes upon EGF stimulation was observed.

• Movie 2 -

  Movie 2. R1-EGFP is endocytosed upon stimulation with EGF. Movie 2 shows a section of Movie 1 with tracks of endocytic vesicles moving from the plasma membrane towards the center of the cell.

• Movie 3 -

  Movie 3. Y163F-R1-EGFP remains associated with the plasma membrane after EGF stimulation. HeLa cells transfected with R1-EGFP were stimulated with EGF and live imaging of the cells was performed with an interval of one frame per second with total of 300 frames. Pinhole settings correspond to an optical thickness z=1.5 μm. The play rate of the movie is 5 frames/second. Y163F mutant was not endocytosed from the plasma membrane upon EGF-stimulation.