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. 2024 Sep 21;300(11):107808. doi: 10.1016/j.jbc.2024.107808

Figure 2.

Figure 2

Vacuoles from elo3Δ cells are fusion impaired.A, time course of fusion for WT or elo3Δ vacuoles. Fusion activity was expressed as the percentage of WT activity. Significance was determined using one-way ANOVA for multiple comparisons for each time point between strains [F (5, 12) = 15.62; ∗∗∗∗p < 0.0001]. Tukey’s multiple comparison test was used for individual p values (n = 3). Error bars represent mean ± SE. ∗∗p < 0.01. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. B, endpoint fusion (90 min) reactions were performed with combinations of WT and elo3Δ (Δ) vacuoles. Significance was determined using one-way ANOVA for multiple comparisons with WT/WT as a control [F (4, 9) = 10.07; ∗∗p = 0.0022]. Dunnett’s test for multiple comparison was used for individual p values. Error bars represent mean ± SE. (n = 3). ∗∗p < 0.01. C, measurement of vacuole diameters after fusion. WT and elo3Δ vacuoles were incubated for the indicated times at 27 °C and images were taken by fluorescence microscopy. Diameters of individual vacuoles in clusters were measured using ImageJ. Data was graphed in a scatter plot of the pooled data. Significance was determined using one-way ANOVA for multiple comparisons [F (7, 1288) = 53.22 p < 0.0001]. Tukey’s test for multiple comparisons was used for individual p values. The bars represent the median with upper and lower quartiles. (n = 3). ∗p < 0.05, ∗∗∗∗p < 0.0001. Scale bar: 5 μm. D, lipid mixing experiments were performed with WT or elo3Δ vacuoles. Fluorescence (ex = 544 nm, em = 590 nm) was measured every 60 s after the addition of ATP. Left, A representative run of the experiment. Right, Quantitation of three experiments calculated by measuring the difference in fluorescence between no ATP and ATP for WT and elo3Δ at the end of the assay. Error bars represent mean ± SE (n = 3). Data was analyzed using an unpaired two-tailed t test ∗∗p < 0.01. E, Western blots of WT and elo3Δ vacuoles that were incubated on ice or at 27 °C for 1 h. Vacuole extracts were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting using antibodies against the specified proteins.