Lys40

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.

Immunochemistry was performed on BoM-MDA-231 control and Runx2 knockdown as previously described [2 (link),17 (link)]. To visualize acetylated microtubules, cells were seeded in 8-well glass chamber slides and incubated overnight. Cells were subjected to control, nocodazole, or vinblastine treatment followed by fixation and imaging. Primary antibodies for α-tubulin and acetylated α-tubulin (Lys40) were obtained from Cell Signaling Technologies (CST) and used at 1:1000 dilutions. Secondary anti-rabbit and anti-mouse antibodies were obtained from Invitrogen and used at 1:1000 dilutions. Anti-rabbit secondary antibody was conjugated with AF-594. Slides were mounted with prolonged antifade reagent with DAPI (Invitrogen, Waltham, MA, USA). Cells were imaged using the Zeiss (LSM70) Confocal microscope equipped with a 63X oil immersion lens. Images were quantified using ImageJ software. Quantification provides integrated density reflecting the intensity of the stain within the region. The data shown provide the mean, and error bars represent the SD.