Figure 1. Rpb3 localizes to centromeres... | Rockefeller University Press

Figure 1.

$Figure 1. Rpb3 localizes to centromeres in a cell cycle–restricted manner. (A and B) Fixed S2 cells immunostained for dCENP-A as a marker for centromeres. Bar, 3 µm. Boxes indicate the 3× enlarged inset (bar, 0.5 µm). (A) Maximum-intensity projection of metaphase cell expressing GFP-Rpb3. Cells were prelysed in PBS/0.1% Triton X-100 for 30 s. (B) Single optical section of cells expressing GFP-Rpb3 elutriated into fractions to enrich for cells in G1 (fractions 1 and 2), S (fraction 3), and G2/M (fractions 4 and 5). The respective FACS profile for each fraction (red) in comparison to nonelutriated cells (blue) is shown in the left column. (C) Graph displaying the correlation between the amount of G1 cells and the cells that showed GFP-positive centromeres in all elutriation fractions, mitotic cells, and asynchronous growing cultures. n = 3 replicates; n = 30–130 cells; data are mean + SD. (D) Graph depicting the presence of the various cell populations in the elutriation fractions and asynchronous growing cultures. Elutriation was performed at 4°C; data were extracted from FACS profiles for each fraction (see also left column of B). n = 3. Data are represented as mean − SD.$

Rpb3 localizes to centromeres in a cell cycle–restricted manner. (A and B) Fixed S2 cells immunostained for dCENP-A as a marker for centromeres. Bar, 3 µm. Boxes indicate the 3× enlarged inset (bar, 0.5 µm). (A) Maximum-intensity projection of metaphase cell expressing GFP-Rpb3. Cells were prelysed in PBS/0.1% Triton X-100 for 30 s. (B) Single optical section of cells expressing GFP-Rpb3 elutriated into fractions to enrich for cells in G1 (fractions 1 and 2), S (fraction 3), and G2/M (fractions 4 and 5). The respective FACS profile for each fraction (red) in comparison to nonelutriated cells (blue) is shown in the left column. (C) Graph displaying the correlation between the amount of G1 cells and the cells that showed GFP-positive centromeres in all elutriation fractions, mitotic cells, and asynchronous growing cultures. n = 3 replicates; n = 30–130 cells; data are mean + SD. (D) Graph depicting the presence of the various cell populations in the elutriation fractions and asynchronous growing cultures. Elutriation was performed at 4°C; data were extracted from FACS profiles for each fraction (see also left column of B). n = 3. Data are represented as mean − SD.

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