Characterization of leep2 knockout cells. (A) Design of the leep2A and leep2B knockout constructs. A blasticidin-resistant cassette (BSR) was inserted to replace part of the open reading frame of leep2A or leep2B via homologous recombination. (B) Knockout clones were confirmed by Southern blotting. Genomic DNA samples were digested with the indicated enzymes and hybridized with the indicated probes. (C) Growth of WT, leep2A⁻, leep2B⁻, and DKO cells on bacterial lawns. Cells were plated clonally with bacteria (Klebsiella aerogenes) on standard medium agar for 5 days. Scale bar, 2 mm. (D) Quantification of phagocytosis of TRITC-labeled yeast in WT and DKO cells. (E) Time-lapse images of yeast phagocytosis in WT cells expressing GFP-Leep2A and Leep2B. White arrowheads indicate macropinocytosis events and yellow arrowheads indicate phagocytosis events. Scale bar, 5 μm. (F) Quantification of TRITC-Dextran uptake in WT and leep2A⁻ cells expressing the indicated constructs. (G) Quantification of TRITC-Dextran uptake in WT and leep2B⁻ cells expressing the indicated constructs. (H) Localization of GFP-Leep2A^N1474K in leep2A⁻ cells. Scale bar, 5 μm. The plot in D shows data points with means and SEM; data were from three independent experiments. The scatter plots in F and G show data points with means and SD; n represents the number of cells analyzed. Significance was determined by one-way ANOVA in F and G. Source data are available for this figure: SourceData FS2.