Tem1-CAAX (green) is targeted away from spindle pole bodies in wild-type cells (this image) but can also still localize to the SPB (CIL# 13881). The CAAX motif from Ras2 targets proteins to the cytoplasmic side of the membrane. tem1Δ::GAL-UPL-TEM1 cells expressing eGFP-TEM1-CAAX from a CEN plasmid were grown on 2% raffinose/2% galactose and transferred to 2% glucose medium prior to image capture. A differential interference contrast (DIC) image is also shown. The tem1Δ::GAL1-UPL-TEM1 strain allows for the rapid, conditional depletion of Tem1. UPL, which stands for ubiquitin-proline-LacI, acts as a destabilizing module that permits rapid degradation of appended proteins. Image is Fig S5D, top panels, in J Cell Biol. (2011) 192: 599-614. Images in Fig S5 include CIL # 13880, 13881.
Cells (MATa tem1::GAL-UPL-TEM1-TRP1 pRS316::eGFP-TEM1-CAAX) 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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