Treatment with sGC Inhibitor ODQ

Protocol Overview

The cells were treated with sGC inhibitor ODQ (10 μM) and the effects of the inhibition of sGC on active TACE and NICD levels were examined. Cells were lysed with the buffer [1% SDS, 10 mmol/L Tris-Cl (pH 7.6), 20 µg/µL aprotinin, 20 µg/µL leupeptin, and 1 mmol/L 4-(2-aminoethyl)benzenosulfonyl fluoride]. The protein concentrations were determined using the Bicinchoninic Acid Protein Assay kit (Pierce, Rockford, IL). Twenty micrograms of protein were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated with the appropriate primary antibody at 4°C overnight. After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour. Proteins were detected with the enhanced chemiluminescence kit (Amersham Pharmacia Biotechnology, Inc., Piscataway, NJ).