690160; Greiner Bio-One) for testing protein expression or at 2.0 �� 104 viable cells in 0.1 ml in 96-well cluster http://www.selleckchem.com/products/Sorafenib-Tosylate.html dishes (no. 3860�C096; Iwaki; Asahi Techno Glass, Chiba, Japan) for the cell viability experiment and were cultured in a humidified atmosphere of 5% CO2 in air at 38 C in an N2-O2-CO2-regulated incubator (no. BNP-110; ESPEC, Osaka, Japan). After 24 h of culture, the medium was replaced with fresh medium containing 0.1% BSA (no. 15408; Roche Diagnostics, Mannheim, Germany), 5 ng/ml sodium selenite (no. S5261; Sigma-Aldrich), 5 ��g/ml transferrin (no. T3400; Sigma-Aldrich), 10 ng/ml LH (USDA-bLH-B6), 100 ��M OP (a specific P4 receptor antagonist; no. ZK98299; Schering AG, Berlin, Germany) and 100 ��M indomethacin (INDO; a COX inhibitor: no. 17378; Sigma-Aldrich).

The doses of LH, OP and INDO were selected based on previous reports [5, 8, 11]. RNA isolation and cDNA synthesis Total RNA was extracted from cultured luteal cells using TRIzol Reagent according to the manufacturer’s directions (no. 15596�C026; Invitrogen, Carlsbad, CA, USA). Total RNA (1 ��g) was reverse transcribed using a ThermoScript RT-PCR System (no. 11146-016; Invitrogen). Quantitative PCR (real-time PCR) Gene expression was determined by real-time PCR using the MyiQ Optical Module (no. 170-9744; Bio-Rad, Tokyo, Japan) and iQ SYBR Green Supermix (no. 170-8880; Bio-Rad) starting with 2 ng of reverse-transcribed total RNA as described previously [22]. Briefly, GAPDH expression was used as an internal control.

For quantification of the mRNA expression levels, the primer length (20 bp) and GC contents of each primer (50�C60%) were synthesized (Table 1) and were chosen using an online software package [23]. PCR was performed under the following conditions: 95 C for 3 min, followed by 45 cycles of 94 C for 15 sec, 55 C for 20 sec and 72 C for 15 sec. Use of the iQ SYBR Green Supermix at elevated temperatures resulted in reliable and sensitive quantification of the RT-PCR products with high linearity (Pearson correlation coefficient r > 0.99). The expression of each gene was evaluated on the basis of the GAPDH expression in the individual samples. Table 1. Primers for real-time PCR Protein analysis Each protein in the cultured bovine luteal cells was detected by Western blotting analysis. The cultured cells were lysed in 30 ��l lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 10% glycerol [no.

G7757; Sigma-Aldrich], Complete [no. 11 697 498 001; Roche Diagnostics, Basel, Switzerland], pH 7.4). Protein concentrations in the Cilengitide lysates were determined by the method of Osnes et al. [24] using BSA as a standard. The proteins samples (50 ��g protein) were then solubilized in SDS gel-loading buffer (50 mM Tris-HCl, 2% SDS [no. 31607�C94; Nacalai Tesque, Kyoto, Japan], 10% glycerol, 1% ��-mercaptoethanol [no. 137-06862; Wako Pure Chemical Industries, Osaka, Japan], pH 6.