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Figure S4. Localization of Sec4 and F-actin in the exo70-38 s3 double mutants. (A) The exo70-38, sec3-201, sec3-203, and exo70-38 s3 cells were grown to early log phase at 25°C and then shifted to the restrictive temperature of 35°C for 2 h. Cells were fixed in formaldehyde and stained with anti-Sec4 polyclonal antibody for immunofluorescence. Sec4 was tightly polarized in single mutants (exo70-38, sec3-201, and sec3-203) but diffused near the buds of double mutants exo70-38 s3-201 (top) and exo70-38 s3-203 (bottom). These results suggest that the secretory vesicles are delivered to the daughter cells; however, these vesicles are not confined to the bud tip for fusion as a result of the loss of the exocyst function at the bud tip membrane. (B) Cells were grown as described in A and then stained with Alexa Fluor 594 Phalloidin. F-actin was mostly polarized in the exo70-38, sec3-201, sec3-203, and exo70-38 s3 cells. Bars, 5 μm.