[PubMed] [Google Scholar]Barth H, Hofmann F, Olenik C, Just I, Aktories K. 2005). Interestingly, some patients homozygous for MK-1775 PLCmutations do not show any signs of the disease (Boyer et al., 2010). PLCenzymes are uniquely regulated by multiple upstream signals including members of the Ras-family and RhoA and generate -like all PLC isozymes- inositol 1,4,5 trisphosphate (IP3) and DAG by cleavage of the phospholipid phosphatidylinositol IGF1 4,5, bisphosphate (PIP2) (reviewed in [Smrcka et al., 2012]). These characteristics of PLCraise the intriguing possibility that increased DAG production after activation of angiotensin receptors by PLCis able to induce TRPC6 activation in podocytes (Dietrich et al., 2010). In summary, there is evidence for an interaction of both proteins or at least for an important role of both components in the same signaling pathway, while recent clinical data indicate that they might work independently from each other. However, while TRPC6 activation by PLC-and PLC-isozymes was extensively MK-1775 studied (Rohacs, 2013), the role of PLCin TRPC activation has never been analysed before (Smrcka et al., 2012). To answer the question if PLCand TRPC6 are members of the same signal transduction cascade which is disturbed in native podocytes in FSGS patients, we set out to characterize TRPC6-PLCinteraction in different cell types utilising different signalling pathways for TRPC6 activation. We now demonstrate that PLCco-immunoprecipitates with TRPC6 in a heterologous expression system and lysates from freshly prepared podocytes. We propose a G12/13, RhoGEF-activation resulting in Rho-mediated PLCstimulation in murine embryonic fibroblasts. Actin stress fiber formation and proliferation however was not altered in PLCdeficient compared to WT podocytes. Our data indicate an important, but redundant activation of TRPC6 by PLCwhich can be replaced by other PLC isoforms. Materials and Methods Animals All animal experiments were approved by the governmental authorities. forward (5-gggatgtcatctcccatcag) and reverse (5-ttgcattcctcctcggatt). Fluorescence intensities were recorded after the extension step at 72 C after each cycle. Samples containing primer dimers were excluded by melting curve analysis and identification of the products by agarose gel electrophoresis. Crossing points were determined by the software program provided by the manufacturer. Relative gene expression was quantified using the formula: (2e[Crossing point GAPDH – Crossing point ]) 100 = % of reference gene expression. MK-1775 In-vitro mutagenesis In vitro site-directed mutagenesis has been used to introduce the M131T point mutation in the murine TRPC6 cDNA. Ten pmol of the forward (5-GTTAACTGTGTGGATTACACGGGCCAGAA TGCCCTACAGC) and the reverse primer (5-GCTGTAGGGC ATTCTGGCCCGTGTAATCCACACAGTTAAC) containing the mutation have been added to the reaction mixtue (5 MK-1775 Phusion HF buffer, 10 l; pcDNA3 plasmid containing the mTRPC6 cDNA, 25 ng; dNTP mix, 10 mM, Phusion HF Polymerase, 2.5 U)(Thermo Scientific, Waltham, MA). PCR was performed using the following conditions: 30 sec initial denaturation and 12 cycles of 30 sec at 95 C, 1 min at 55 C, 9 min at 86 C. Then 1 l (10 u) was added to the PCR reaction to digest parental methylated and hemimethylated DNA before transformation of E. DH5. Plasmid MK-1775 DNA was isolated from single colonies on ampicillin-containing agar plates and sequenced. For incorporation of the TRPC6M131T cDNA into the lentiviral vector pWPXL the insert was released from pcDNA3 by digestion with and (Thermo Scientific, Waltham, MA) and then ligated into the and restriction sites of pWPXL. Co-immuno precipitation Cells from two cell culture dishes were washed with PBS and 2 ml of lysis buffer (20 mM Tris-HCL, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 1% SDS, 5 mM EDTA) was applied for 30 min at 4 C. Proteins were homogenized, 200 ng of antibody (GFP ab.: Takara Bio Europe SAS, Saint-Germain-en-Laye, France, #632377; Hemagglutinin ab.: Sigma, Taufkirchen, Germany, #H6903; IgG from mouse serum: Sigma, Taufkirchen, Germany, #I5381; PLC1 ab.: Sigma, Taufkirchen, Germany, #SAB2104946; PLCab.: Santa Cruz Biotechnology Inc., Heidelberg,.