Tem1 (green) localizes to spindle pole bodies (SPB) during metaphase. Control image for CIL# 13876 in which Tem1 loading onto SPBs was severely affected in bfa1∆ cells. DAPI (blue) and DIC (hidden) are also shown. CIL# 13871 and 13870 show corresponding anaphase images. Image is Fig S4B, top panels, in J Cell Biol. (2011) 192: 599-614. Images in Fig S4 include CIL# 13877, 13876, 13879, 13878.
Cells (MATa tem1::TEM1-yEGFP::SpHIS5) were fixed in 2.5% formaldehyde for 10 min, washed twice, and resuspended in 0.1 M potassium phosphate buffer, pH 6.4. Cells were then fixed for 10 min in 80% ethanol and resuspended in 1 mg/ml DAPI. Imaging was performed at 25C using a Leica DM6000 microscope equipped with a 100x/1.40 NA oil immersion objective lens, A4, L5, and TX2 filters, and a digital CCD camera (DFC350, Leica). Pictures were processed with LAS AF (Leica) and ImageJ software.
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