Comparison of SidK-AL568 and V₁A antibody specificity. (A and B) Demonstration of the Costes thresholding method used before colocalization analyses. HeLa cells were stained with SidK-AL568 (A; magenta) or α-V₁A (B; magenta), and the trans-Golgi was immunostained as described in Materials and methods. For both A and B, the left panel shows staining of the magenta channel before Costes thresholding, while the right panel shows the same channel after thresholding. Outlines of cells are indicated by dotted lines. Scale bars: 5 µm. Images in A and B correspond to Fig. 4, C and H, respectively. (C) Immunoblotting of the indicated protein amounts of yeast membranes, rat membranes, and HeLa and RAW264.7 lysates for the V-ATPase using the V₁A antibody, α-ATP6V1A. The V₁A band is marked with an open arrowhead. V₁A blot is shown in comparison to its GAPDH loading control. (D) Immunoblotting of 20 µg protein samples of HeLa and RAW264.7 lysates with the V₁A antibody. The V₁A band is marked with an open arrowhead. V₁A blot is shown in comparison to its GAPDH loading control. Blots in C and D are representative of three similar experiments. Source data are available for this figure: SourceData FS2.