Phosphatase inhibitor mixture 1

AQP cRNA-injected and water-injected oocytes were lysed in 200 μl of breaking buffer (50 mM Tris, pH 7.4; 1% IGEPAL; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM phenylmethylsulfonyl fluoride; 1 mM protease inhibitor mixture; 1 mM phosphatase inhibitor mixture 1, all purchased from Sigma-Aldrich Co. Oocyte protein extracts were resolved on 7.5% gradient sodium dodecyl sulfate–polyacrylamide gels and electrotransferred to polyvinylidene difluoride membranes by the use of Trans-Blot SD semi-dry electrophoretic transfer cell (Bio-Rad, Hercules, CA). The membranes were blocked overnight in Starting Block T20 Blocking buffer (Thermo scientific, Waltham, MA) at 4°C. The membranes were washed with TBS and incubated in blocking buffer containing 1:1000 dilution mouse anti-myc tag monoclonal antibody (Cell Biolabs Inc., San Diego, CA) at room temperature for 1 hr. After extensive washing with TBS, the membranes were incubated with an alkaline phosphatase-labeled secondary antibody (Millipore, Billerica, MA) in blocking buffer for 2 hrs at room temperature. The bands were visualized using 1 step NBT/BCIP suppressor (Thermo Scientific, Waltham, MA). This procedure was similar to one previously described46 (link).