Fig. 4. Localisation of flp 1p. (A) Living cells expressing flp 1p-GFP were photographed, and images were processed as described in Materials and Methods. (B) Cells expressing flp 1p-GFP were fixed and processed for indirect immunofluorescence. DNA was stained with DAPI and flp 1p-GFP was detected with rabbit antiserum to GFP, followed by FITC-conjugated goat-anti rabbit serum. (C) Cells expressing flp 1p-HA were fixed and processed for indirect immunofluorescence. DNA was stained with DAPI; flp 1p-HA was detected with 12CA5 followed by CY3-conjugated goat anti-mouse serum; DNA was stained with DAPI; and the nucleolar antigen fibrillarin was detected using affinity-purified rabbit antibodies against S. cerevisiae nop 1p followed by FITC-conjugated goat-anti-rabbit serum. Note that while the nucleolar staining overlaps in the merge, the dot corresponding to the spindle pole body does not. (D) Cells expressing flp 1p-HA were fixed and processed for indirect immunofluorescence. DNA was stained with DAPI; the spindle pole antigen sad 1p was detected with rabbit anti-sad 1p, followed by FITC-conjugated goat anti-rabbit serum; and flp 1p-HA was detected with 12CA5, followed by CY3-conjugated goat anti-mouse serum. (E) nda3-KM311 cells expressing flp 1p-GFP were incubated for 5 hours at 20°C, fixed and processed for indirect immunofluorescence. flp 1p-GFP was detected with rabbit antiserum to GFP followed by FITC-conjugated goat anti-rabbit serum, while F-actin was detected using Rhodamine-conjugated Phalloidin. (F) nda3-KM311 cells expressing flp 1p-GFP were incubated for 5 hours at 20°C, fixed and processed for indirect immunofluorescence. flp 1p-GFP was detected with rabbit antiserum to GFP followed by FITC-conjugated goat anti-rabbit serum, while DNA was detected using DAPI. (G) Living cells expressing flp 1p-GFP were treated with Latrunculin A for 10 minutes, then photographed. Images were processed as described in Materials and Methods. i indicates an interphase cell in image 1; the arrow indicates the location of the spindle pole body in the interphase cell. Image 2 shows the disorganised dot-like staining observed in mitotic cells, and image 3 shows a pair of interphase cells that have been treated with LatA. Note that the nucleolar and spindle pole body staining are both still present.