Rat ip3 enzyme linked immunosorbent assay kit

The concentration of IP3 in the three cell types was determined using a rat IP3 enzyme-linked immunosorbent assay kit (Cusabio Biotech Co., Ltd., Wuhan, China) according to the manufacturer's protocol. Following treatment with the five candidates, cells (2x10 7 ) were harvested and homogenized in ice-cold phosphate-buffered saline (pH 7.2-7.4) with a glass homogenizer (Sigma-Aldrich). Total cell lysates were prepared with a glass homogenizer and PBS, and centrifuged at 5,000 x g for 5 min at room temperature, following which the supernatant was collected for analysis. An anti-IP3 detection antibody was added and samples were incubated at 37˚C for 60 min, followed by the addition of substrate solution for 15 min at 37˚C. The reaction was terminated by the addition of stop solution and the plates were read at an absorbance of 450 nm using a VersaMax Plate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).
Statistical analysis. Statistical analyses were performed using SPSS software version 10.10 (SPSS, Inc. Chicago, IL, USA).
One-way analysis of variance, followed by Tukey's post hoc test, was performed to identify significant differences between the vehicle and candidate-treated groups, or between the untreated and Lop-treated groups. All values are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.