Figure 3.

AURKA is targeted by SPOP in a reciprocal crosstalk. (A) SPOP inversely regulates AURKA protein levels in C4-2 cells. (B) Histogram shows decrease in AURKA level with SPOP overexpression. The data presented as mean ± SEM obtained from three independent experiments. * p < 0.05 vs. C4-2 control cells. (C) SPOP knockdown increases AURKA in C4-2 cells. (D) Histogram shows increase in AURKA levels with SPOP knockdown. The data are presented as mean ± SEM obtained from three independent experiments. * p < 0.05 vs. C4-2 control cells. (E) and (F) 22Rv1 cells showing change in AURKA protein levels with SPOP overexpression. (G) and (H) 22Rv1 cells showing change in AURKA protein levels after SPOP silencing. (I) SPOP decreases the half-life of AURKA. C4-2, and SPOP-shRNA-C4-2 were treated with 10 μM cycloheximide and protein lysates were collected at the indicated times for western blot analysis. (J) AURKA protein levels was quantified and plotted relative to the level at t = 0. (K) SPOP augments AURKA degradation in 22Rv1 cells. (L) Graphical representation of AURKA degradation rate in cells treated as in Figure 3K. (M) SPOP increases AURKA degradation by promoting its ubiquitylation. C4-2 cells were co-infected with 6x-His-ubiquitin (6x-His-Ub) along with SPOP retrovirus for 30 h, followed by MG132 treatment for 12 h. AURKA was immunoprecipitated and ubiquitylation analyzed using 6x-His antibody. Each experiment was done at least three independent times. Representative data are shown.