SidK-AL568 specifically labels the V-ATPase of mammalian and yeast cells. (A) HeLa cells were stained with SidK-AL568 (magenta) either directly (left) or after incubation with a fivefold excess of unlabeled SidK_1-278 (right). F-actin labeled with fluorescent phalloidin (blue). (B) Spheroplasts of wild-type or vma1Δ S. cerevisiae were stained with SidK-AL568 (magenta) as detailed in Materials and methods. Nuclei labeled with DAPI (cyan). Outlines of spheroplasts, visualized by differential interference contrast microscopy, are indicated by dotted lines. (C and D) HeLa cells transfected with V_Oa2- (C) or V_Oa3-GFP (D) constructs (green) were stained using SidK-AL568 (magenta). Nuclei labeled with DAPI (blue). Cell outlines indicated by dotted lines. Side panels show individual SidK-AL568 and V_Oa channels. Colocalization of SidK-AL568 and the corresponding V_Oa subunit is shown in orange, and the Mander’s coefficient (M) between the subunit and SidK-AL568 is indicated. (A–D) Compressions of confocal stacks representative of ≥30 fields from three or more experiments. Scale bars: 5 µm.