Cos 7 crl 1651

The candidate compounds identified through SBDS were purchased from ChemBridge (San Diego, CA, USA). Gene constructs for expression of Grb14 (hGrb14/pcDNA3.1D/V5-His-TOPO) and IR (pFN21AE3464) in mammalian cells are reported previously [24 (link)]. Rabbit anti-IRS-1 and anti-phospho-IRS-1 (Tyr608) mouse/(Tyr612) human antibodies were purchased from Merck (Whitehouse Station, NJ, USA). Horseradish peroxidase (HRP)-conjugated anti-His-tag monoclonal antibody was purchased from MLB (Nagoya, Japan). HRP-conjugated anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody was purchased from FUJIFILM Wako Pure Chemical Industries (Osaka, Japan). HRP-conjugated anti-IRβ mouse monoclonal antibody and goat HRP-conjugated anti-rabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HEK293T(CRL-3216) and COS-7(CRL-1651) were purchased from American Type Culture Collection (Manassas, VA, USA). Other chemicals of reagent grade were obtained from Sigma, FUJIFILM Wako Pure Chemical Industries, and Nacalai Tesque (Kyoto, Japan).