Fig. 8. Alfy binds PtdIns(3)P in vitro and colocalizes partially with PtdIns(3)P in vivo. (A) Cytosol from HeLa cells was incubated with liposomes containing an equal mixture of phosphatidylserine and phosphatidylinositol, in the presence [PtdIns(3)P] or absence (control) of 5% PtdIns(3)P, included at the expense of phosphatidylinositol. Liposomes were collected by centrifugation and the cytosolic supernatant (S, black) and the liposome pellet (P, grey) were analysed for the presence of Alfy by SDS-PAGE (7.5%) and immunoblotting using the anti-Alfy antibody. Error bars denote s.e.m. from three experiments. (B) Nitrocellulose strips containing the indicated lipids were incubated with GST, GST fused with a tandem FYVE domain from Hrs or GST fused with the FYVE domain of Alfy, and analysed for protein binding. The lipids spotted in column 1 in each strip are indicated to the left, whereas lipids spotted in column 2 are indicated to the right. (C) HeLa cells grown on coverslips were starved for 45 minutes in HBSS, permeabilized, fixed and stained against endogenous Alfy (left), and the presence of PtdIns(3)P detected with GST-2xFYVE-Alexa 488 (middle). The merged image (right) shows that some of the Alfy-positive structures (arrows and inset) colocalized partially with PtdIns(3)P-containing areas in the cells. Scale bar, 10 µm.