Mouse anti at1r

Following the contractile studies, the SV rings were flash frozen in liquid nitrogen and stored at −80 °C for Western blot analysis. Frozen SV rings were crushed with manual mill SK-200 (Tokken, inc.) and suspended in transmembrane protein extraction buffer 1 (ProteoExtract Transmembrane Protein Extraction Kit; Calbiochem-Novabiochem). Preparation for the membrane fractions and Western blot analysis was performed as described previously [11] . Equal amounts of protein (5.5 μg per lane) were separated by SDS-10% polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane Hybond-P (GE Healthcare Japan). The membrane was preincubated with 1% skim milk in Tween-Tris-buffered saline (10 mM Tris–HCl [pH 7.4], 150 mM NaCl, and 0.1% Tween-20) and reacted with rabbit anti-5-HT_2A receptor (1:2000, Santa Cruz Biotechnology), rabbit anti-β-actin (1:2000, Cell Signaling Technology) or mouse anti-AT₁R (1:1000, abcam) overnight at 4 °C in Immuno-enhancer Reagent A (Wako). The immunoreactive bands were reacted with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies in Immuno-enhancer Reagent B (Wako), visualized using the Immuno-Star enhanced chemiluminescent detection system (Wako), and quantified using a luminoimage LAS-4000 analyzer (GE Healthcare Japan). Protein levels of 5-HT_2A receptor and AT₁R were normalized by the level of β-actin.