Skip to main content
. 2010 Sep;1(6):1–8. doi: 10.1038/ncomms1061

Figure 2. Intracellular calcium release is sufficient for AurA activation.

Figure 2

(a) HEK293 cells overexpressing AurA were incubated with 5 μM thapsigargin for the indicated periods of time, and then cell lysates were analysed by western blot. Graph shown below indicates ratio of phAurA to total AurA. (b) AurA immunoprecipitation and in vitro kinase assay, after thapsigargin treatment. HEK293 cells overexpressing AurA were treated with 5 μM thapsigargin or control dimethylsulpfoxide (DMSO) for 5 min, AurA was immunoprecipitated and incubated with histone H3 substrate in an in vitro kinase assay. phHH3, antibody to phosphorylated histone H3. *P<0.05. (c) Left, an experiment similar to that shown in a, except that it is performed in the presence of 50 μM BAPTA-AM or control DMSO. Right, HEK293 cells overexpressing AurA were incubated with 20 μM BAPTA-AM (30 min) plus 7.5 mM EGTA (2 min) before the addition of 5 μM thapsigargin for the indicated periods of time, with analysis as for a. (d) An experiment similar to that shown in a, except that it is performed in the presence of 5 mM EGTA alone for the indicated periods of time (right panel) or in the presence of 5 mM EGTA (2 min) before the addition of 5 μM thapsigargin. (e) HEK293 cells overexpressing AurA were incubated with 5 μM thapsigargin and indicated concentration of ionomycin for the indicated periods of time, and then cell lysates were analysed by western blot using indicated antibodies. (f) HK2 cells were treated with DMSO, 5 μM thapsigargin (Tg) or 0.5 μM ionomycin (Ion), and then T288-phAurA levels were analysed by enzyme-linked immunosorbent assay (ELISA), based on A450 nm. To confirm the specificity of ELISA signal, a parallel group of HK2 cells were pre-treated with the AurA inhibitor PHA680632 (PHA) at 500 nM concentration. *P=0.05. Lysis buffer was used as a negative control. (g) AurA-overexpressing HEK293 cells were transfected with siRNA-depleting NEDD9 (siNEDD9) or scrambled control siRNA (scr), and then were incubated with 5-μm thapsigargin and processed as in a. For this and subsequent SDS–PAGE analyses, each experiment was performed three times independently, with error bars indicating the standard error. *P<0.05.