Figure EV5. Measuring the effects on microtubule stability after NSC23766 administration in Rcc2 or Anax2 siRNA‐treated ECs.

Top: β3WT endothelial cells were transfected with control pool (CP), Anxa2 or Rcc2 smart pool siRNA and allowed to recover for 48 h. Cells were then adhered to fibronectin‐coated coverslips for 60 min at 37°C before treated with DMSO (veh) or 50 μM NSC23766 (+) and incubated at 37°C for a further 15 min. Coverslips were moved to ice for 15 min. Soluble tubulin was then washed out using PEM buffer before fixing with −20°C methanol. Immunostaining was carried out for alpha‐tubulin to allow counting of the number of cold‐stable microtubules per cell. Bars = mean (±SEM) number of microtubules per cell shown as a percentage relative to the CP/veh control (n = 100 cells per condition, from two independent experiments). Scale bar = 5 μm.