Hrp anti mouse

For IP experiments, HEK293FT cells were transfected with 500 ng each of YAP and nV5-TEAD4 cDNA constructs, and lysed in RIPA buffer (derived from 10x stock (Millipore, Billerica, MA); final concentration of components: 50 mM TRIS.HCl pH 7.2, 120 mM NaCl, 1% Nonidet P40 (v/v), 1 mM EDTA and 0.1% (v/v) SDS; supplemented with 2 mM sodium orthovanadate, 6 mg/ml sodium pyrophosphate and PhosSTOP and Protease Inhibitor Cocktail (both from Roche, Switzerland)) 48 hr afterwards. Lysates (250 µg) were then incubated with YAP1 antibody overnight under rotation at 4°C, followed by incubation with Dynabeads Protein G (Invitrogen, Carlsbad, CA) for 2 hr under rotation at 4°C. Immunoprecipitates were washed three times with RIPA buffer lacking SDS, eluted with Laemmli Sample Buffer (BioRad, Hercules, CA) by incubation at 95°C for 5 min and resolved by standard SDS-PAGE gel electrophoresis and Western Blotting. The following antibodies were used. For IP: YAP1 (EP1674Y; Abcam (United Kingdom),ab52771). For Western Blot analysis: YAP1 (D8H1X XP; Cell Signaling Technology (Danvers, MA), #14074) and V5 (Invitrogen (Carlsbad, CA), R96025) as primary antibodies; HRP-anti-rabbit (Cell Signaling Technology (Danvers, MA), #7074) and HRP anti-mouse ( GE Healthcare (United Kingdom), NA931V) as secondary antibodies.

Total cell lysates from human rhabdomyosarcoma cell lines and human myoblasts were obtained following lysis in 2% SDS lysis buffer supplemented with protease inhibitors (Santa Cruz Biotechnology). Western blot analysis used primary antibodies: rabbit a-NOTCH1 (Abcam), a-Cleaved NOTCH1 (Cell Signaling Technology), a-SNAI1 goat pAB (R&D Systems), a-Myosin Heavy Chain mouse mAb (monoclonal antibody) myosin heavy chain (a-MF20, R&D), MEF2C rabbit mAb (CST); and secondary antibodies: HRP (horseradish peroxidase) anti-rabbit (CST, 7074) or HRP anti-mouse (GE Healthcare, NA93IV). Membranes were developed using an ECL reagent (Western Lightning Plus ECL, PerkinElmer; or sensitive SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific). Membranes were striped, rinsed, and re-probed with the respective internal control rabbit a-Lamin B1 (Abcam) or rabbit a-GAPDH (CST).