Yol1 34

ECs cultured on glass-bottom chamber slides or EC sprouts in 3-D culture were fixed with 2% paraformaldehyde/PBS for 10 min or 30 min, respectively, permeabilized in 0.1% Triton X-100/PBS, and blocked in 10% goat serum/PBS before immunostaining. Cells were stained with α-tubulin (Invitrogen, YOL1/34, diluted 1:500), acetylated-α-tubulin (Lys40) (Cell Signaling Technologies, #5335, diluted 1:250), delta 2-tubulin (Novusbio, NB100-57397, diluted 1:100), detyrosinated α-tubulin (Biolegend, 909503, diluted 1:500), EB1 (Santa Cruz, sc-47704, diluted 1:200), or PODXL (ThermoFisher Scientific, PA5-28116, diluted 1:200) antibody or phalloidin (Invitrogen, A12379, diluted 1:50). ECs and EC sprouts were analyzed by epi-fluorescence (Nikon Eclipse 90i) and confocal (Nikon A1R) microscopy. For quantitative analyses of post-translationally modified and total α-tubulins, the positive area for each immunostaining was determined in each cell by Volocity^® software. Total of 15 cells from three different experiments were examined.

Cells were grown to mid-log phase at the permissive temperature (23°C) and arrested in G1 with 10 μg/ml alpha-factor for three hours (with a second equivalent amount added after two hours). Cells were then released into medium without alpha-factor at the restrictive temperature (30°C) to inactivate ycg1-2. Cells were collected at 60-, 75-, and 90-minutes following release to enrich for anaphase cells. Samples were fixed and stained as previously described [61 (link)], with minor modifications. Permeabilized cells were stained with anti-tubulin (YOL1/34, Invitrogen) and anti-Nop1p primary antibodies (Encor) for two hours, and anti-rat FITC (Jackson Immunoresearch) and goat anti-mouse Alexafluor 594 (Invitrogen) secondary antibodies for two hours, both at room temperature. Cells were imaged on a Nikon Eclipse E400 microscope. To ensure that cells at similar stages of anaphase were being compared, late anaphase cells were identified as those with an elongated spindle and clearly segregated DAPI masses. Nop1 segregation was then scored as equal or unequal (either absent or uneven) in only late anaphase cells. A minimum of 100 cells per strain were scored in each experiment.