Identification of PP6 as the Aurora A T-loop phosphatase. (A) HeLa cells were transfected with siRNA pools for 72 human phosphatase subunits for 54 h, treated for 18 h with nocodazole, and then lysed in the absence of phosphatase inhibitors for 2 h on ice. Control samples were lysed in the presence or absence of phosphatase inhibitors (PP-Inh). Samples were Western blotted for total Aurora A and Aurora A pT288. The graph shows the ratio of phosphorylated/total Aurora A (n = 2). The red line indicates the median value for Aurora A phosphorylation. (B) HeLa cells were transfected with siRNA pools for 73 human phosphatase subunits for 54 h, treated for 18 h with nocodazole, and then lysed in the absence of phosphatase inhibitors for 2 h on ice. In addition, control and PPP6C-depleted samples were lysed in the presence or absence of phosphatase inhibitors. All samples were analyzed on Mn²⁺ Phos-tag gels to better resolve phosphorylated from nonphosphorylated Aurora A and then Western blotted for total Aurora A and actin as a loading control. Red boxes highlight siRNA 44 (PPP6C) in which Aurora A phosphorylation is retained and an adjacent sample in which it is not. (C–E) HeLa cells were transfected for 48 h with control, PPP6C si08, SAPS1–3, PP1C A, PP1C B, PP1C C, and PP2C A siRNA duplexes. Lysates were then prepared from these cells in immunoprecipitation buffer containing or lacking phosphatase inhibitor cocktail and Western blotted. Graphs show phosphorylated Aurora A levels (n = 3). Molecular mass is given in kilodaltons. Error bars indicate the standard error of the mean. Where present, the red and black lines indicate the phosphorylated and nonphosphorylated forms of Aurora A. White lines indicate that intervening lanes have been spliced out.