Launch visionworksls

Western blot (WB) and immunoprecipitation (IP)/immunoblotting analyses were performed on whole cell lysates and nuclear fractions according to published methods [19 (link),20 (link)]. The anti-phospho-c-Abl (Tyr245), anti-β-catenin, anti-14-3-3 (pan), anti-phospho-14-3-3 (Ser186) and anti-Sumo 1 antibodies were purchased from Cell Signaling Technology. The anti-phospho-JNK (Thr 183) was purchased from Merck Millipore. The anti-CBY1 was previously described [16 (link)]. The anti-β-actin antibody used as loading control was purchased from Santa Cruz Biotechnology. The anti-histone H1 used as control for nuclear protein loading was purchased from Genetex. Signal intensities in single blots obtained in three separate experiments were measured by means of ChemiDoc-It instrument (UVP) equipped with a dedicated software (Launch VisionWorksLS from Euroclone). The differences among signal intensities were evaluated for statistical significance using the paired Student’s t-test.

AML cells were collected by centrifugation, washed with PBS 1% Phenylmethanesulfonyl fluoride PMSF (Sigma-Aldrich) and total protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK), and then subjected to Western blotting. Membranes were saturated for 1 hour at room temperature in blocking buffer (1X tris-buffered saline, 5 M NaCl, 20 mM Tris-HCl; pH 8.0, 0.1% Tween-20, 4% BSA) and then incubated overnight at 4°C with the specific primary antibodies: rabbit anti-pAkt Thr308 [1:3000] (18F.H11; Abcam) or p21 [1:1000] (#2947, Cell Signaling) in 4% BSA in TTBS (TBS 0,1% Tween-20) for 15–20 h at 4°C. Membranes were washed three times for 5 min in TTBS and secondary antibodies (donkey anti-rabbit HRP (sc-2313), donkey anti-mouse HRP (sc-2314) [1:40000] (Santa Cruz Biotecnology) were added for 1h at room temperature, and then membrane-bound were washed three times for 5 min in TTBS as described previously. Signal intensities in single blots were measured by means of ChemiDoc-It instrument equipped with a dedicated software (Launch VIsionWorksLS, Euroclone). Protein expression was quantified by band densitometric analysis using IMAGEJ 1.44p Launcher software (National Institutes of Health, Bethesda, MD, USA).

Western blot (WB) and immunoprecipitation /immunoblotting (IP/IB) analyses were performed on whole cell lysates and nuclear fractions according to published methods [23 , 24 (link)]. The anti-β-catenin, anti-PLK1, anti-phospho-PLK1 (Thr210), anti-AURKA, anti-phospho-AURKA (Thr288) and anti-GADD45α (D17E8) were purchased from Cell Signaling Technology. The anti-FOXM1 was purchased from Abcam. The anti-β-actin antibody used as loading control was purchased from Santa Cruz Biotechnology. Signal intensities in single blots obtained in three separate experiments were measured by means of ChemiDoc-It instrument (UVP) equipped with a dedicated software (Launch VisionWorksLS from Euroclone). The differences among signal intensities were evaluated for statistical significance using the paired Student’s t-test.