Supplemental Materials

This article contains the following supporting material:

• Supplemental Figure 2 - Clathrin localization and transferrin uptake are unchanged by RNAi.
  (A) Ser-W3 cells (1x10⁴/coverslip) were first treated with siRNA for amphiphysin 1 and cultured for 24 hours. Control cells (upper panels) and siRNA treated cells (lower panels) were fixed, permeabilized and stained with anti-amphiphysin 1 antibodies (mab 3) (left) and anti clathrin heavy chain antibodies (Santacruz Biotech., CA) (middle). Bar: 10 μm.
  (B) Transferrin uptake assay was performed according to Asa et al (Asa et al., Mol. Biol. Cell, 15, 1666-1679, 2004). Briefly, siRNA treated Ser-W3 cells (closed square) or control cells (open circle) were incubated at 4C° for 1hr with human biotinylated-transferrin (2 μg/ml; Sigma, MO). Cells were then washed and moved to 31C° water bath for various time period (0, 5, 10, 15 min) to arrow transferrin internalization. To measure internalized transferrin, surface-bound transferrin was stripped by washing with acidic buffer at 4C° , and cells were lysed. The amount of biotinylated transferrin in the lysate was determined by ELISA as described in Buss et al (Buss et al., EMBO J., 20, 3676-3684, 2001). Internalized transferrin was calculated as percentage of total surface-bound transferrin using data from two independent experiments.
• Supplemental Figure 3 - Transfection of amphiphysin 1 cDNA rescues PS-stimulated ruffle formation in amphiphysin 1 knocked down cells.
  (A) Ser-W3 cells were first treated with siRNA for amphiphysin 1 and cultured for 24 hours. Cells (1x10⁴/coverslip) were then transfected with 1 μg of cDNA of amphiphysin 1 cloned into pIRES2-AcGFP1 expression vector (Clontech, CA). After 24 hours, the cells were stimulated with 0.25 mM PS liposomes at 37 °C for 10 min. F-actin is visualized by phalloidin staining. Ruffles were formed in GFP- and amphiphysin 1-positive cells (arrowhead). Bar: 10 μm.
  (B) The number of GFP- and ruffle-positive cells were determined and expressed as a percentage of the total number of cells analyzed (right). Amphiphysin 1 siRNA treated cells were used as negative control (left). Thirty cells in 10 independent fields were counted in each experiment. All results represent the mean ± SEM. from three experiments.
• Supplemental Figure 1 - Amphiphysin 1 accumulates at phagosomes.
  TM4 cells (1x10⁴/coverslip), mouse Sertoli cell line, were transfected with amphiphysin 1-myc as described in Figure 5. After 24 hours of transfection, the cells were incubated with PS beads at 37 °C for 180 minutes. To visualize attached beads to cell surface, cells were incubated with streptavidin-Rhodamine (Pierce Biotech., IL), which specifically binds to biotinylated phosphatidylethanolamine (PE) (Invitrogen, CA), at 4 °C for 30 minutes before fixation. They were fixed, permeabilized with digitonin and stained with anti-myc antibodies (upper panels) and Alexa488-phalloidin (lower panels). Arrowheads indicate the incorporated beads, which are avidin-negative, surrounded by amphiphysin 1 and polymerized actin. Bar: 10 μm.