Anti clu sc 5289

Cells were fixed with 100% methanol for 5 minutes at room temperature. After blocking with 3% bovine serum albumin, the cells were incubated with anti‐SREBF1 (14088‐1AP) (Proteintech, IL) or anti‐CLU (sc‐5289) (Santa Cruz Biotechnology, Dallas, TX) overnight at 4°C. The cells were washed with phosphate‐buffered saline, and incubated with Alexa Fluor 594‐labeled anti‐rabbit (1:1,000) secondary antibodies (Invitrogen; Thermo Fisher Scientific) for 60 minutes at room temperature. Samples were analyzed by fluorescence microscope system (KEYENCE, Osaka, Japan), and the fluorescence intensity was analyzed using the BZ‐X Analyzer.

ESCs were collected with Pierce IP Lysis Buffer (Pierce, Waltham, MA, USA) supplemented with protease and phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. A total of 0.5 mg of proteins were incubated with 1–4 μg of anti-HSP90 (ab13492, Abcam, UK), anti-phospho-AKT (4060, Cell Signaling Technology, Danvers, MA, USA), anti-CLU (sc-5289, Santa Cruz, Dallas TX, USA), or IgG (CS200621, Millipore, Burlington, MA, USA) antibodies at 4°C overnight and then with PureProteome Protein A/G Magnetic Beads (Millipore, Burlington, MA, USA) at room temperature for 30 min. The immunoprecipitates were washed three times with 0.1% Tween-20 in PBS and then subjected to immunoblotting with the indicated antibodies.

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).