Anti rabbit igg horseradish peroxidase

For Western blotting 20ug of exosome or cell lysate protein were analyzed by 10% SDS PAGE. Protein concentration was determined using a microBCA kit (Pierce) as per the manufacturer’s recommendations. Alix (3A9) (Cell Signaling Technology, Cat # 2171, 1:1000 dilution), GAPDH (Santa Cruz Biotechnology, Cat # sc-32233, 1:200 dilution), Snail (C15D3) (Cell Signaling Technology, Cat # 3879, 1:1000 dilution), ZEB1 (H-102) (Santa Cruz Biotechnology, Cat #: Sc-25388, 1:200 dilution), and Beta-Tubulin (Cell Signaling Technology, Cat # 2128, 1:1000 dilution). Anti-Rabbit IgG-horseradish peroxidase, anti-goat IgG-horseradish peroxidase and anti-mouse IgG-horseradish peroxidase were used as secondary antibodies (Santa Cruz Biotechnology). Chemiluminescent detection was performed using Super Signal West Femto Maximum Sensitivity Substrate (Thermo Scientific) as per the manufacturer’s recommendations. Image capture was performed using a ChemiDoc imaging system (Bio-Rad).

Total protein was extracted from cells using a RIPA cell lysis buffer containing protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific). The total amount of protein was quantified in RIPA extracts using the BCA kit. Equivalent quantities of RIPA-solubilized proteins were resolved by 7–14% SDS/polyacrylamide gels, and the separated proteins were transferred to nitrocellulose membranes (Merck Millipore, MA, USA). Membranes were blocked with 5% non-fat milk in Tris Buffered Saline with 0.1% Tween^® 20 (TBST) and probed with the following primary antibodies: anti-YAP, anti-phospho-YAP Ser127 (pYAP), anti-CDK4, anti-CDK6 (all from Cell Signaling Technology) and anti-α-tubulin (Santa Cruz) diluted 1 : 1000 in 5% BSA in TBST overnight at 4°C. Primary antibody-probed blots were visualized with anti-rabbit IgG-horseradish peroxidase (Santa Cruz) diluted 1 : 5000 in 5% BSA in TBST for 1 h at room temperature. Protein detection was performed by autoradiography using enhanced chemoluminescence (Merck Millipore). Films were scanned and saved as digital images, and the intensities of protein bands were analysed using ImageJ software (National Institutes of Health, MD, USA). The relative level of protein, normalized by the loading control α-tubulin level, is expressed as mean fold-change of control (1.0) ± s.d. from three independent experiments.