Quasi-irreversible engagement of AURKA in cells. (a) COS-7 cells were transfected with WT or K162Q (#) Flag-AURKA and treated with probe 3 (30 min). After lysis in the presence of sodium borohydride and TAMRA-azide conjugation, samples were analyzed by in-gel fluorescence and western blotting. (b) Concentration-dependent labeling of Flag-AURKA (~50 kDa TAMRA band, mean ± SD, n=3). (c,d) COS-7 cells were transfected with Flag-AURKA and treated with probe 3 (2 μM, 30 min), followed by washout for the indicated times. Samples were analyzed as described above (mean ± SD, n=3). * and NT indicate non-transfected cells.