“…After transfection, cells were treated with or without various pharmacological inhibitors such as LY294002 for PI3K, PD98059 for mitogen-activated protein/ extracellular signal-regulated kinase 1, rapamycin for mechanistic target of rapamycin-mediated S6 kinase, staurosporine for broad-spectrum protein kinases, H89 for protein kinase A and PP2 for Src-family tyrosine kinases. Afterward, the cells were lyzed in cell lysis buffer (Chang et al, 2006), and the protein concentration was quantified using the BCA assay (Thermo Fisher). The lysates were resolved in 7.5-10% polyacrylamide gel containing sodium dodecyl sulfate, transferred to nitrocellulose membranes, and probed with purified CA502 ascitic antibody (32 mg/ml) or antibody specific to Myc tag, GST-tag (Thermo Fisher), p21 Waf/Cip1 , p27 Kip , cyclin Ds, cyclin E, pTyr 416 -Src, c-Src (Cell Signaling Technology, Beverly, MA, USA) or SIK3.…”