Fig. 4. Subcellular localization of Meu14-GFP in fixed cells. (A) Detection of Meu14-GFP protein by western blotting, which is expressed in a meiosis-specific manner. A band corresponding to Meu14-GFP with the expected size (shown by an arrowhead) is detected predominantly 4-8 hours after nitrogen starvation, after which it suddenly disappears and cannot be detected at all at 10 hours and 12 hours (uppermost panels). In contrast, a band for GFP alone is detected primarily at 6-12 hours (middle panels), probably because the Meu14 portion of Meu14-GFP is easily degraded, releasing the GFP portion. Western blot probed by an anti-Cdc2 antibody was used as a loading control. Asterisks indicate nonspecific bands. The extracts of wild-type cells (TP4D-5A/TP4D-1D) transformed with pRGT81-meu14⁺ (meu14⁺-GFP) or pRGT81 (GFP vector) are used to identify the expressed Meu14-GFP protein. These extracts are denoted by 14 or G, respectively (rightmost lanes). (B) Fluorescence from Meu14-GFP observed at various stages of meiosis. Cells were immunostained with the TAT1 antibody to mark the microtubules (red). Cells bearing meu14⁺-gfp (YDO120) were induced to enter meiosis and then chemically fixed and analyzed by fluorescence microscopy for DNA (blue), Meu14-GFP (green), and microtubules (red). The three images are merged and shown in the rightmost panels. (C) Correlation of the distance between the two Meu14-GFP rings (c) with either (a) the distance between two nuclei or (b) the length of the microtubules. (D) meu14⁺-gfp (YDO120) cells immunostained by the anti-Sad1 antibody to visualize the SPB at various stages of meiosis. It appears that Meu14-GFP is located on the cytoplasmic side of the SPB (white arrow). (E) meu14⁺-gfp (YDO120) cells immunostained by the anti-NPC antibody mAB414. Bar, 10 µm.