Cleaved caspase 3 antibody 9661

For detection of sCLU, cytoplasmic components were isolated using a LysoPure Nuclear and Cytoplasmic Extractor kit (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), and nuclear components were discarded. Cytoplasmic lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), and the proteins were transferred onto polyvinylidene fluoride membranes. Blots were incubated with respective primary antibodies overnight at 4°C, followed by incubation with corresponding secondary antibodies for 1 hour. Proteins were visualized by enhanced chemiluminescence using EzWestLumi Plus and Ez‐Capture MG (ATTO Corp., Tokyo, Japan). Anti‐CLU (sc‐5289) and anti‐GAPDH (sc‐32233) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Anti‐p‐Akt (4060), anti‐pan Akt (2920), anti‐p‐Erk (1/2) (9106), and anti–phosphorylated mammalian target of rapamycin (p‐mTOR) (5536) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti‐Erk (1/2) antibody (MAB1576) was purchased from R&D Systems (Minneapolis, MN). Cleaved Caspase‐3 antibody (#9661) was purchased from Cell Signaling Technology (Danvers, MA).