P stat5ay694

SDS-PAGE and western blots were performed following the standard protocols [31 (link)]. Antibody binding to bands was detected using an ECL detection system (Sage Creation). The primary antibodies used were specific to STAT5A (1:1000, #4807, Cell Signaling Technology, Danvers, MA, USA), DGCR8 (1:1000, #6914, Cell Signaling Technology), p-STAT5A^Y694 (1:1000, #4322, Cell Signaling Technology), p-STAT3^Y705 (1:1000, #9145, Cell Signaling Technology), PCNA (1:1000, #13110, Cell Signaling Technology), GAPDH (1:10000, ab181602, Abcam, Cambridge, UK).

To identify the relative levels of phosphorylation at the 46 kinase phosphorylation sites, the Proteome Profiler Human PhosphoKinase Array Kit (ARY003B, R&D Systems) was used in the DLD1 and HT29 cells transfected with the control and CIP2A-specific shRNAs according to the manufacturer’s instructions. The resulting spots were identified and images were quantified using the Image lab software (Bio-Rad) and Microsoft Excel software. The proteins were evaluated through western blotting, which were identified as follows: p53 (Abcam), p-p53 (S392) (Abcam), STAT5a (Cell Signaling, USA), p-STAT5a (Y694) (Cell Signaling, USA), Cyclin D1 (Cell Signaling, USA), Bax (Cell Signaling, USA), p21 (Cell Signaling, USA), p-AKT (T308) (Cell Signaling, USA), AKT (Cell Signaling, USA), p-ERK1/2 (Cell Signaling, USA), ERK1/2 (Cell Signaling, USA), and β-actin (Santa Cruz).