As shown in Figure 5A, ERK and JNK were activated while mTOR was repressed

To detect dominant negative TBRII expres sion, an anti HA tag antibody was used. Antibodies TBRI, TBRII, and TBRIII were used to detect endogenous TBRI, TBRII, and TBRIII expres sion, respectively. A mouse monoclonal anti FLAG anti supplier ABT-888 body was used to detect FLAG C2 ATF2 expression. To verify equivalent loading, membranes were subsequently incubated with an actin antibody. siRNA knockdown To suppress mRNA levels of Smad2 3, ATF2, TAK1, TRAF6, RhoA, TBRI, TBRII, and TBRIII, we performed knockdown experiments with small interfering RNA. The ON TARGET plus SMART pool siRNAs and negative control siRNA were obtained from Dhar macon. VSMCs were plated on 60 mm dish and after overnight incubation, cells were trans fected with 20 nM of negative control or target siRNA in Opti MEM I Reduced Serum Medium using Lipofectamine RNAiMAX as described by the manufac turers protocol.<br><br> Assessment of RhoA activation RhoA activation was assessed using an Active Rho Pull Down and Detection Kit by immunoprecipitating purchaseAfatinib GTP bound RhoA with GST fu sion protein of Rhotekin RhoA binding domain. Briefly, VSMCs plated in 150 mm dish were washed with ice cold PBS and lysed in buffer contain ing 25 mM Tris pH 7. 2, 150 mM NaCl, 5 mM MgCl2, 1% NP 40, and 5% glycerol. Cell lysates were incu bated with 400 ug of GST Rhotekin RBD and glutathione resin at 4 C for 1 h. Following washing, bound Rho was eluted by SDS sample buffer. The eluted samples and the total cell lysates were then subjected to Western blot ana lysis with RhoA antibody to detect active and total RhoA, respectively.<br><br> Quantitative real time PCR Total RNA was isolated using an RNeasy Mini kit according to the manufacturers instructions. One ug RNA was first reverse transcribed to cDNA with random primers using SuperScript III reverse supplier AG-1478 transcriptase. Quantitative real time PCR was performed with the transcribed cDNA and SYBR FAST Universal 2X qPCR Master Mix in triplicates using the 7500 real time PCR system to detect CRP2 mRNA expression. Postnatal cardiomyocytes have a limited proliferation rate that does not suffice to replenish the CM that are mas sively lost after Myocardial Infarction. During human life span approximately half of the cardiomyocytes are replaced. This indicates that there is a significant level of physiological proliferation of cardiomyocytes.<br><br> Thus, novel therapies that promote the proliferation of CM after acute Myocardial Infarction may alleviate post infarct complications such as heart failure. Over the past decade, mesenchymal stem cells emerged as promising candidates for cardiac therapy. Stem cells and progenitor cells from sources that vary from bone marrow to adipose tissue and the heart itself have shown to be beneficial in animal models of aMI and in clinical trials. The current dogma is that stem cells act primarily through paracrine intervention in the damaged cardiac microenvironment i. e. through secretion of trophic factors. The secretion profile and the fate of administrated cells change upon a host microenvironment. Current research on preconditioning BM MSC with the hypoxic and the inflammatory fac tors found in post MI microenvironment improve the cardioprotective outcome of the therapeutic cells.