Immunofluorescence Staining of Tiam1

For IF, cells were grown on coverslips and fixed with 100% ice-cold methanol for 5 min at −20°C. Cells were washed and then blocked in 1% BSA in PBS (v/v) for 1 h, before successive incubation with primary antibodies (overnight at 4°C) and then secondary antibodies (1 h at room temperature). Coverslips were mounted onto glass slides using Fluromount-G (Southern Biotech) along with Hoechst 33342 (1:5000) for nuclear staining, or a droplet of ProLong Gold anti-fade reagent containing the DNA stain DAPI. Staining of endogenous Tiam1 was performed using a protocol described by Whalley et al. (2015) (link), which was shown to specifically detect centrosomal Tiam1.