Rabbit anti nox 1

To obtain protein extracts, cells were lysed with 50 mM Tris buffer (pH 8.0) containing 0.2 mM EDTA, 5% SDS and 50 mM DTT for 20 min at 99 °C and centrifuged at 20,000 × g, 20 min, 4 °C. The supernatants were collected, and total protein concentration was determined using the Bradford method. 10 μg of proteins per extract or coimmunoprecipitation reaction were resolved by 10% SDS-PAGE, transferred to a nitrocellulose membrane using wet transfer and stained with Ponceau S to test the quality of the transfer. Membranes were blocked for 1 h with 3% BSA in PBS and then incubated overnight at 4 °C with primary antibodies (rabbit anti-ALDOA, 1:1000, Sigma; rabbit anti NOX-1, 1:3000, Sigma) diluted in PBS. The membranes were then incubated for 1 h in RT with secondary antibodies (goat anti-rabbit IgG-HC, HRP conjugated, 1:1000, Sigma) diluted in PBS. Rabbit anti-β-actin (1:3,000, Sigma) and IgG heavy chains were used as a loading control in experiments with, respectively, cellular extracts and coimmunoprecipitation. A peroxidase substrate, 3,3’-diaminobenzidine (DAB), was used to develop a color reaction.