Immunofluorescence

U2OS cells were fixed with cold methanol (–20°C) for 4 min or 0.1–0.25% gluteraldehyde + 4% paraformaldehyde (Electron Microscopy Sciences) for 10 min. Autofluorescence was quenched by 0.1% sodium borohydride (Sigma-Aldrich) after aldehyde fixation and cells permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for another 10 min. Tyrosinated α-tubulin and detyrosinated α-tubulin were immunostained using rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:100–500, Bio-Rad) and rabbit polyclonal anti-detyrosinated α-tubulin (1:250–2,000; Liao et al., 2019), respectively. Other primary antibodies used were human anti-centromere antibodies (ACA, 1:100–2,000, kind gift from B. Earnshaw, Welcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK, or S. Geley). GFP-tagged constructs were visualized by means of direct fluorescence. Alexa Fluor 488 (1:200–2,000, Themofisher), STAR-580, and STAR-RED (1:200, Abberior) were used as secondary antibodies, and DNA was counterstained with 1 µg/ml DAPI (Sigma-Aldrich).