Fig. 6

Increased levels of FYVE and ATG8L proteins and numbers of their vesicles by nocodazole, DTT, and G418 treatment. A, B, C Nocodazole, DTT, and G418 treatment increased the level of FYVE protein. The pPFYVE stable transfectants were cultured in growth medium containing (A) 5 μM nocodazole, (B) 5 mM DTT, and (C) 217 μM G418, or the same volume of solvent (H2O or Me2SO) for 24 h and then subjected to SDS-PAGE and Western blot analysis. The blot was probed with anti-HA and anti-RAN antibodies, respectively. SDS-PAGE with Coomassie Blue staining is included as a control for equal protein loading. D The band intensity from triplicate Western blots of FYVE experiments was quantified using Image J as described in Fig. 1E. *, p < 0.05. E FYVE-localized vesicles can be induced by nocodazole, DTT, and G418 treatment. The pPFYVE stable transfectants were cultured in growth medium with 5 μM nocodazole, 5 mM DTT, and 217 μM G418, or the same volume of solvent (H2O or Me2SO) for 24 h and then subjected to immunofluorescence assay using anti-HA antibody for detection. Quantification of FYVE-localized vesicles in nocodazole, DTT, and G418 treated cells during vegetative stage was performed using Imaris software. **, p < 0.01 (n = 200–300 cells/condition). F, G, H Nocodazole, DTT, and G418 treatment increased the level of ATG8L protein. The pPTUATG8L stable transfectants were treated with (F) nocodazole, (G) DTT, and (H) G418 and then subjected to SDS-PAGE and Western blot analysis. The blot was probed with anti-HA and anti-RAN antibodies, respectively. SDS-PAGE with Coomassie Blue staining is included as a control for equal protein loading. I The band intensity from triplicate Western blots of ATG8L experiments was quantified using Image J as described in Fig. 1E. *, p < 0.05. J ATG8L-localized vesicles can be induced by nocodazole, DTT, and G418 treatment. The pPTUATG8L stable transfectants were treated with nocodazole, DTT, and G418 and then subjected to immunofluorescence assay using anti-HA antibody for detection. Quantification of ATG8L-localized vesicles in nocodazole, DTT, and G418 treated cells during vegetative stage was performed using Imaris software. *, p < 0.05. **, p < 0.01 (n = 200–300 cells/condition)