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. 2014 Aug 18;289(39):26937–26948. doi: 10.1074/jbc.M114.595058

FIGURE 5.

FIGURE 5.

The amount of enzyme homomers and heteromers after their BFA- or nocodazole-induced re-localization into the ER or to the Golgi mini-stacks, respectively. A, immunofluorescence micrograph showing the accumulation of the depicted FRET enzyme constructs in the ER in BFA-treated cells as determined by their co-localization with the ER marker protein (PDI). Scale bar, 10 μm. B, FRET microscopy quantification of the amount of enzyme homomers and heteromers in the ER after BFA treatment. Note that the drug does not markedly reduce the amount of enzyme homomers in the ER (left, dark columns) relative to the Golgi-localized homomers in untreated control cells (light columns). In contrast, the amount of heteromers is significantly (by 50–70%) reduced in the drug-treated cells. The results are presented as percentages of untreated controls (mean ± S.D., n = 8). C, relocalization of the depicted FRET enzyme constructs upon nocodazole (NDZ, 1 μg/ml) treatment or after a 4-h recovery period. Note that the enzymes accumulate in the Golgi mini-stacks after nocodazole treatment and relocalize back to the juxtanuclear Golgi structure after removal of the drug. D–F, FRET microscopy measurements in cells expressing the depicted enzyme homomers or heteromers either before or after nocodazole treatment or after the 4-h recovery period. The results are presented as percentages of untreated controls (mean ± S.D., n = 10; *, p < 0.05; **, p < 0.01; ***, p < 0.001). D, enzyme homomers. Note that the drug-induced relocalization into the ER does not affect the amount of enzyme homomers when compared with that of the Golgi-localized homomers in untreated cells. E, enzyme heteromers. Note the marked reduction in the amount of enzyme heteromers after their accumulation in the Golgi mini-stacks in nocodazole-treated cells and their reappearance after the 4-h recovery period. F, FRET measurements with the GnT-I or GalT-I homomers in the presence of an interacting HA-tagged heteromeric enzyme construct. Note the marked increase in the amount of enzyme homomers in nocodazole-treated cells and the decrease after the 4-h recovery period.