Stage-specific differential association of Brr6 with mitotic SPBs. (A) Antibodies raised and purified against a equimolar mixture of the first 79 and last 44 amino acids of the Brr6 recognized a single band at 34 kD on blots of cell extracts prepared from wild-type cells (lane 2; brr6⁺). This band was replaced by a band at 37 kD and 65 kD in strains in which three Pk epitopes or a single EGFP had been inserted at the start of the brr6⁺ ORF (lanes 3 [brr6.NEGFP] and 4 [brr6.N3Pk]) and was supplemented by a band at 37 kD when a Brr6.3Pk fusion protein was expressed in wild-type cells (lane 1; leu1::ura4⁺nmt41brr6N3Pk). (B) Size selected mid-log phase cells were inoculated at a density of 10⁶ cells/ml, and samples were processed for calcofluor staining to score septa (graph) or anti-Brr6 Western blot to monitor Brr6 protein. The sets of one and two asterisks above the blot in B show the peaks of maximal septation in the first and second cell cycles, respectively, as indicated on the graph. (C–E) Anti-Brr6 immunofluorescence microscopy of mid-log phase cells reveals punctate staining around the nuclear periphery and association with the SPBs of mitotic cells. The intensity of the signal varies at different stages of mitosis. Cells are numbered in descending order of brightness in a particular field. The intensity of the two metaphase SPBs in cell 2 (D) is markedly lower than that in the earlier mitotic stage in cell 1 in the same field, which indicates that the amount of Brr6 associating with SPBs diminishes at metaphase. Similarly, the reduced level of staining in the early and late anaphase cells 2 and 3 (C) shows that Brr6 association with the SPB peaks in mid-anaphase (cell 1) at a point before the nuclear envelope can define two distinct yet connected spheres (cell 3). At the earliest stages of mitosis, Brr6-stained SPBs appear to reside within the area defined by the nuclear peripheral staining (asterisks in C and E). Bars, 5 µm.