Figure 5.
Figure 5. UNC50 mediates recruitment of GBF1 to the Golgi apparatus. (A) WT cells were transfected with control or anti-GBF1 siRNAs. After 48 h, cells were imaged to detect GM130 and GBF1. (B) Quantification of GBF1 fluorescence intensities from A. Intensity in cells transfected with control siRNA was normalized to 100 (n = 15 cells). *, P < 0.05 by t test. (C) Trafficking of His-tagged STx2B was assayed in cells transfected with control or anti-GBF1 siRNAs for 48 h. Cells were fixed 60 min after start of transport. STx2B was detected using a polyclonal antibody against the His tag. Golgi was demarcated using a monoclonal antibody against GM130. (D) Quantification of the Pearson’s coefficient for colocalization between STx2B and GM130 from C (n = 15 cells). *, P < 0.05 by t test. (E) WT or ΔUNC50 cells were fixed, and endogenous GM130 and GBF1 were detected using monoclonal and polyclonal antibodies, respectively. (F) Quantification of GBF1 levels from E. Intensity in WT cells was normalized to 100 (n = 15 cells). (G) Quantification of the relative intracellular distribution of GBF1 from E. Signal for GM130 was used to delineate the Golgi (n = 25 cells). *, P < 0.05 between indicated groups using one-way ANOVA and Tukey-Kramer post hoc test. (H) ΔUNC50 cells were transfected with CRISPR-sensitive myc-UNC50 for 24 h. After this, cultures were fixed and stained to detect endogenous GBF1, GRASP65, and the myc tag. Asterisks denote transfected cells. (I) Quantification of GBF1 levels from H. Intensity in untransfected cells was normalized to 100 (n = 15 cells). (J) Quantification of the relative intracellular distribution of GBF1 from H. Golgi was demarcated using the signal for GRASP65 (n = 15 cells). *, P < 0.05 between indicated groups using one-way ANOVA and Tukey-Kramer post hoc test. (K) WT cells were transfected with HA-tagged dominant-negative ARF1T31N for 24 h. After this, transport of His-STx2B was assayed. Cells were fixed 60 min after start of transport. ARF1 was detected using a monoclonal antibody against the HA tag, STx2B was detected using a polyclonal antibody against the His tag, and GRASP65 was detected using a polyclonal antibody. Asterisks denote transfected cells. Bars, 25 µm. (L) Quantification of the Pearson’s coefficient for colocalization between STx2B and GRASP65 from K (n = 15 cells). *, P < 0.05 by t test; error bars show means ± SEM.

UNC50 mediates recruitment of GBF1 to the Golgi apparatus. (A) WT cells were transfected with control or anti-GBF1 siRNAs. After 48 h, cells were imaged to detect GM130 and GBF1. (B) Quantification of GBF1 fluorescence intensities from A. Intensity in cells transfected with control siRNA was normalized to 100 (n = 15 cells). *, P < 0.05 by t test. (C) Trafficking of His-tagged STx2B was assayed in cells transfected with control or anti-GBF1 siRNAs for 48 h. Cells were fixed 60 min after start of transport. STx2B was detected using a polyclonal antibody against the His tag. Golgi was demarcated using a monoclonal antibody against GM130. (D) Quantification of the Pearson’s coefficient for colocalization between STx2B and GM130 from C (n = 15 cells). *, P < 0.05 by t test. (E) WT or ΔUNC50 cells were fixed, and endogenous GM130 and GBF1 were detected using monoclonal and polyclonal antibodies, respectively. (F) Quantification of GBF1 levels from E. Intensity in WT cells was normalized to 100 (n = 15 cells). (G) Quantification of the relative intracellular distribution of GBF1 from E. Signal for GM130 was used to delineate the Golgi (n = 25 cells). *, P < 0.05 between indicated groups using one-way ANOVA and Tukey-Kramer post hoc test. (H) ΔUNC50 cells were transfected with CRISPR-sensitive myc-UNC50 for 24 h. After this, cultures were fixed and stained to detect endogenous GBF1, GRASP65, and the myc tag. Asterisks denote transfected cells. (I) Quantification of GBF1 levels from H. Intensity in untransfected cells was normalized to 100 (n = 15 cells). (J) Quantification of the relative intracellular distribution of GBF1 from H. Golgi was demarcated using the signal for GRASP65 (n = 15 cells). *, P < 0.05 between indicated groups using one-way ANOVA and Tukey-Kramer post hoc test. (K) WT cells were transfected with HA-tagged dominant-negative ARF1T31N for 24 h. After this, transport of His-STx2B was assayed. Cells were fixed 60 min after start of transport. ARF1 was detected using a monoclonal antibody against the HA tag, STx2B was detected using a polyclonal antibody against the His tag, and GRASP65 was detected using a polyclonal antibody. Asterisks denote transfected cells. Bars, 25 µm. (L) Quantification of the Pearson’s coefficient for colocalization between STx2B and GRASP65 from K (n = 15 cells). *, P < 0.05 by t test; error bars show means ± SEM.

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