Supplemental Material

This article contains the following supporting material:

• Figure 1 - Transferrin recycling in PLD1 and PLD2 depleted cells. Cells were loaded with Alexa 488-transferrin as in Figure 2 and allowed to release transferrin for 1 hr at 37°C (A) or for 30 minutes at 32°C (B). siPLD2# indicates a different pair of siPLD2 oligonucleotides not used to obtain the data shown in Figure 2. Representative experiments are shown.
• Figure 2 - PLD2 overexpression did not increase transferrin recycling. HeLa cells were transfected with plasmids encoding GFP or cotransfected with GFP and HA-PLD2 plasmids at a ratio of 1 to 5. In the second set of samples essentially all cells expressing GFP also expressed PLD2 (A). Transferrin recycling using Alexa 633-transferrin was measured as in Figure 6 measuring only GFP positive cells in both control (GFP) and PLD2 overexpressing (GFP:PLD2) cell populations (B). PLD activity was also measured as in Figure 4 (C).
• Figure 3 - Control and siPLD2 cells were immunolabeled with panPLD, Rab11, EEA1, CD63 and GM130 antibodies. Cells were fixed with 3.7 % formaldehyde except those labeled with PLD2 were fixed with methanol.