Mutations in Syt VII palmitoylation sites cause defects in lysosomal targeting and BMM phagocytosis. (A) Schematic model of Syt VII depicting in red the three cysteines (C35, C38, and C41) adjacent to the transmembrane domain (highlighted in yellow) that were mutated to serines in Syt VII (C/S)–YFP. (B) Phagocytosis levels were quantified in Syt VII^−/− BMMs transduced with adenovirus expressing Syt VII–YFP, Syt VII (D/N)–YFP, and Syt VII (C/S)–YFP after exposure to 25 zymosan particles per cell for 1 h. The reduced phagocytosis that is observed in Syt VII^−/− BMMs was rescued by wild-type Syt VII–YFP, but not by Syt VII (D/N)–YFP (the Ca²⁺-binding mutant) or Syt VII (C/S)–YFP (the palmitoylation mutant). The asterisk indicates statistically significant differences from the control construct Syt VII–YFP (Student’s t test, P < 0.05). The data represent the means, and the error bars represent the standard deviation of triplicate determinations. (C) Western blot of the transduced Syt VII^−/− BMM cell lysates with anti-GFP antibodies showing comparable amounts of expressed Syt VII–YFP, Syt VII (D/N)–YFP, or Syt VII (C/S)–YFP. The bottom panel shows the loading control, which was probed with antiactin antibodies. The values on top reflect the ratio of Syt VII/actin quantified by ImageJ software. (D) Live confocal images of Syt VII^−/− BMMs transduced with adenovirus encoding Syt VII–YFP or Syt VII (C/S)–YFP and Lamp1-RFP. Syt VII–YFP colocalizes with Lamp1 on tubular lysosomes, but Syt VII (C/S)–YFP does not. Bars, 6 µm.