Mouse lrh1

TLR-3 cells were plated in collagen-coated 24-well plates and transfected with 200 ng cAMP reponse element-luciferase (CRE-luciferase) reporter (Qiagen, Valencia, CA), 150 ng β-galactosidase, 200 ng mouse LRH-1, and 200 ng ATF2 (Addgene). 48 hr later, the cells were treated with TM or the following kinase inhibitors: 10 μM D-JNKi for JNKs (Sigma), 1 μM SB202190 for p38 (Tocris), 10 μM GW84362X for PLK1/PKL3 (Tocris, Bristol, UK), or 1 μM GSK650394A for SGK (Tocris). 24 hr after treatment, cells were lysed in Tropix lysis buffer (100 mM potassium phosphate, 0.2% TritonX-100, pH 7.8) plus DTT. Lysates were plated in triplicates in 96-well plates and 85 μl of reaction buffer was automatically injected by the luminometer. Reaction buffer for each well was prepared as follows: 0.7 μl galacton (Applied Biosystems, Foster City, CA), 88 μM luciferin, 2.4 mM ATP, and 11.9 mM MgCl₂ in 0.11M Tris-phosphate buffer, pH 7.8. Firefly luciferase activity was measured and samples were incubated 1 hr at room temperature. 100 μl Tropix Accelerator (Applied Biosystems) was then automatically injected and measured to quantify β-galactosidase activity, which was used to normalize luciferase activity values.