The efficiency of ER-α siRNA transfection (0.05–0.5 μM of lyophilized siRNA) was determined by real time RT-PCR from cDNA samples Selleck Bcl-2 inhibitor isolated from mouse primary mesangial cells. The transfection efficiency (40%) was determined by comparing the relative mRNA expression of ER-α in scrambled and ER-α siRNA-transfected mesangial cells. For this purpose, we used primers for mouse ER-α: forward 5′-ATGAAAGGCGGCATACGGAAAG-3′, reverse 5′-CACCCATTTCATTTCGGCCTTC-3′ (Fig. 4). Primary mesangial cells were incubated with Pam3CsK4 for a period of 6 h. Total

RNA was isolated from mesangial cells using TRIzol (Invitrogen, USA) and the Qiagen RNA isolation kit (Qiagen, USA). The cDNA samples were prepared by following the instructions of the Superarray cDNA preparation kit (Superarray, USA). Amplification of cDNA was performed by quantitative

real time PCR (qRT-PCR) (MyIQ BioRad, USA) using RT2 real-time SYBR Green fluorescein PCR mastermix from Superarray (USA). The real time primers used were mouse MCP1: forward 5′-CTTCTGGGCCTGCTGTTCAC-3′, reverse 5′-GGGATCATCTTGCTGGTGAA-3′; and mouse β-actin: forward 5′-TCCTCCCTGGAGAAGAGCTA-3′ and reverse 5′-CCAGACAGCCACTGTGTTGGC-3′. The relative mRNA expression of MCP1 was determined by comparison Ruxolitinib mw with the expression of the housekeeping gene mouse β-actin as well as with corresponding unstimulated controls (control value set as 1). We found similar expression patterns with 18S RNA primers for real time

PCR (SABiosciences, Qiagen, USA) as for constitutive β-actin. The results were expressed as the mean ± SE of three different experiments. The student’s t-test was used to determine statistical significance of the results compared with corresponding controls. The significance level (p <0.05) was determined by calculated p-values. Phosphorylation at Serine104/106 and Serine 118 on the ER-α protein determines ER-α activity [30,31] and is required for estrogen-mediated gene expression. However, a relationship between ER-α activation and TLR2 agonist-induced MCP1 production in activated mesangial cells is not yet well determined. We wanted to determine Tryptophan synthase the effect of ER-α and phosphorylated ER-α (Serine118) on TLR2-mediated induction of MCP1 production in mesangial cells. The 4–6 weeks old female MRL/lpr and C57BL/6 mice utilized were phenotypically normal and healthy. These mice were used to isolate mesangial cells. The results presented in Fig. 1 demonstrated that the TLR2 agonist lipoteichoic acid (LTA) induced ER-α activation (Fig. 1A). The fluorescent intensity of MRL/lpr mesangial cells following treatment with LTA in vitro was found to increase compared to the untreated control. These observations ( Fig. 1A) also indicated an overall activation of ER-α in mesangial cells. The effect of LTA treatment on localization of phosphorylated ER-α [pER-α (Serine 118)] in MRL/lpr mesangial cells was determined in vitro. The results presented in Fig.