Figure 11.
The BCAS3-C16orf70 complex binds to PtdIns3P in vitro. (A) CBB staining of purified GFP-His, GFP-WIPI1-His, GFP-BCAS3-His/GFP-C16orf70-His and GFP-BCAS3R401A-His/GFP-C16orf70-His. (B) Schematic diagram of the liposome flotation assay. After incubating the liposome solution with purified proteins, OptiPrep was added to a final concentration of 35% and then 30% OptiPrep and buffer were sequentially layered on top. After centrifugation, the Bottom (B), Middle (M) and Top (T) fractions are collected and analyzed by western blotting. (C) Flotation assays using the purified proteins and liposomes containing the indicated phosphoinositides were performed. GFP-His and GFP-WIPI1-His were detected with an anti-GFP antibody, and GFP-BCAS3-His was detected with an anti-BCAS3 antibody. (D) Flotation assays using liposomes with various concentrations of PtdIns3P were performed. GFP-BCAS3-His was detected with an anti-BCAS3 antibody