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Insulin/IGF1 receptor hybrid activation assay by immunoprecipitation and Western blot analysis was performed by addition of 30 μl of Protein G-Agarose (Roche Diagnostic, Indianapolis, IN, USA) in 300 μl buffer (100 mM Hepes, pH 7.8, 100 mM NaCl, 10 mM MgSO 4 , 0.02% Tween-20) and 1 μg of the appropriate antibody for 3 hours at 4°C followed by brief centrifugation

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BACTERIOSTATIC WATER:10ml

According to Holden et al