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Figure S1. Response of three PI(3)P-binding proteins to starvation. (A) HEK-293 cells were transfected with plasmids encoding GFP-DFCP1, GFP-FYVE domain from FENS-1, or GFP-PX domain from p40 PHOX. 36 h after transfection, the cells were left untreated (control) or starved for 45 min as indicated. Note that all three proteins translocated to punctate structures upon starvation, but the nature (and number) of these structures were distinct. (B) HEK-293 cells were cotransfected with plasmids encoding RFP-MAP-LC3 and GFP-tagged DFCP1, FYVE domain, or PX domain as indicated. 36 h after transfection, the cells were starved for 45 min, and the extent of colocalization between MAP-LC3 and the three proteins was examined. Note that overexpression of DFCP1 reduced the amount of MAP-LC3–containing autophagosomes, but that those that were formed colocalized with DFCP1. There was less colocalization of MAP-LC3 with the FYVE and PX domain constructs. Overall, 50% of GFP-DFCP1–containing puncta colocalized with MAP-LC3, whereas the corresponding values for GFP-FYVE and GFP-PX were 6 and 4%, respectively. Images on the bottom show enlarged views of the boxed regions. Bars, 20 µm.