UNC50 is required for the optimal transport of STx1 to the Golgi. (A) Viability of WT or ΔUNC50 cells exposed to indicated amounts of STx1 holotoxin for 16 h was assessed as described in Fig. 3 A (n = 3). (B) Depiction of the mean LD₅₀ of STx1 from A with 95% confidence intervals (CIs). *, P < 0.05 by t test. (C) Trafficking of Alexa Fluor 555–labeled STx1B was assayed in WT or ΔUNC50 cells. Cells were fixed at 0 or 60 min after start of transport. Golgi was demarcated using a monoclonal antibody against giantin. (D) Quantification of the Pearson’s coefficient for colocalization between STx1B and giantin from C (n = 25 cells per group). *, P < 0.05 for the comparison between WT cells at 60 min and ΔUNC50 cells at 60 min by one-way ANOVA and Tukey-Kramer post hoc test. (E) Quantification of STx1B fluorescence intensities from C. Intensity of WT cells at 0 min was set to 100. Intensities of other groups were expressed relative to WT at 0 min (n = 25 cells per group). *, P < 0.05 for the difference between WT cells at 0 min and other groups using one-way ANOVA and Dunnett’s post hoc test. (F) ΔUNC50 cells were treated with leupeptin (100 µg/ml) and pepstatin (50 µg/ml) for 24 h or left untreated. Transport of fluorescently labeled STx1B was then assayed. Cells were fixed at 0 or 60 min after start of transport (0-min time point is not depicted). Golgi was demarcated using a monoclonal antibody against giantin. (G) Quantification of STx1B fluorescence intensities from F. For each treatment condition, intensity at 0 min was normalized to 100, and intensity at 60 min was expressed relative to 0 min (n ≥ 25 cells per group). *, P < 0.05 by t test. (H) Immunoblot analyses were performed using lysates prepared from WT or ΔUNC50 cells to detect GPP130, using a polyclonal antibody, or tubulin, using a monoclonal antibody. (I) Quantification of GPP130 levels normalized to tubulin from H (n = 3). *, P < 0.05 by t test. (J) ΔUNC50 cells were treated with or without leupeptin (100 µg/ml) and pepstatin (50 µg/ml) for 24 h and stained to detect GPP130, using a monoclonal antibody, and giantin, using a polyclonal antibody. Arrowheads show GPP130 in cytoplasmic punctae. Dotted boxes mark insets. (K) Quantification of GPP130 levels from J. Note that images for WT cells are not depicted in J. For quantification, fluorescence intensity in WT cells was set to 100, and intensities in ΔUNC50 cells were expressed relative to WT (n ≥ 25 cells per group). *, P < 0.05 for the difference in GPP130 levels between indicated groups using one-way ANOVA and Tukey-Kramer post hoc test. (L) ΔUNC50 cells were transfected with CRISPR-sensitive myc-tagged UNC50. 1 d after transfection, cells were fixed and imaged to detect GPP130, using a polyclonal antibody, and the myc tag. Asterisks denote transfected cells. (M) Quantification of GPP130 levels from L. Note that images for WT cells, which were not transfected with the myc-UNC50 plasmid, are not depicted in L. For quantification, fluorescence intensity in WT cells was set to 100, and intensities in ΔUNC50 cells were expressed relative to WT (n ≥ 15 cells per group). *, P < 0.05 for the difference between WT and other groups using one-way ANOVA and Dunnett’s post hoc test. (N) ΔUNC50 cells were transfected with CRISPR-sensitive myc-tagged UNC50. 1 d after transfection, transport of fluorescently labeled STx1B was assayed. Cells were fixed at 0 or 60 min after start of transport (the 0-min time point is not depicted) and imaged to detect STx1B and myc. Bars: (C, F, J [main images], L, and N) 25 µm; (J, insets) 5 µm. (O) Quantification of STx1B fluorescence intensities from N. For each transfection condition, intensity at 0 min was normalized to 100, and intensity at 60 min was expressed relative to 0 min (n = 15 cells per group). *, P < 0.05 by t test; error bars show means ± SEM.