Horseradish peroxidase conjugated goat anti mouse immunoglobulin g igg

Protein was extracted from tissues and cells using RIPA lysis buffer containing proteinase inhibitor (Sigma-Aldrich, Otsu, Japan). The protein concentration was determined using the BCA Protein Assay Kit (Vigorous Biotechnology Beijing, Beijing, China). Equal amounts of protein lysates (20 μg each lane) were resolved using 10% SDS-PAGE gels and then electroblotted onto nitrocellulose membranes (Millipore, Madison, WI, USA). The membranes were blocked for 2 hr with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20, and incubated at 4°C overnight with the following primary antibodies: mouse monoclonal anti-human IRS-1 (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-human URI (1:500, Santa Cruz Biotechnology), and mouse monoclonal anti-human GAPDH (1:5,000, Santa Cruz Biotechnology). GAPDH was used as an internal control for protein loading. The membrane was further incubated with horseradish-peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) (1:5,000, Santa Cruz Biotechnology) for 1 hr at room temperature. The immune complexes were detected by enhanced chemiluminescence (ECL; Cell Signaling Technology, Danvers, MA, USA). The integrated density of the band was quantified by Quantity One software (Bio-Rad, Hercules, CA, USA).