Project description:S11537: anti-FLAG beads incubated with mouse B cell extract
S11538: FLAG-Pacs1 purified from 293T cells on anti-FLAG beads
S11536: FLAG-Pacs1 purified from 293T cells on anti-FLAG beads incubated with mouse B cell extract.
Project description:We report the results of ChIP-Seq experiment investigating the MtrA TCS response regulator. The experiment utilised an in trans copy of mtrA with an N-terminal 3xFlag tag encoded on the plasmid pMS82 (containing a phiBT1 integration site). The experiment utilised beads associated with anti-flag antibodies, specific for the Flag tagged protein. We report important targets involved in cell division and salt stress with key findings supporting the hypothesis that MtrA is essential. Samples were taken over a time course of 10h, 12h, 14h, 16h, 18h and 20h.
Project description:We report the results of ChIP-Seq experiment investigating the Rrf2 family transciptional regulator RsrR (putatively name Redox sensitive response regulator). The experiment utilized an in trans copy of rsrR with an N-terminal 3xFlag tag encoded on the plasmid pMS82 (containing a phiBT1 integration site). The experiment utilized beads associated with anti-flag antibodies, specific for the Flag tagged protein and a WT host strain. We report >600 target binding sites for the RsrR regulon encompassing a broad range of functional targets with specific reference to those associate with NADPH and NADH metabolism and synthesis.
Project description:Obesity and diabetes are well known risk factors for nonalcoholic fatty liver disease (NAFLD), but the genetic factors contributing to the development of NAFLD remain poorly understood. Here we describe two semi-dominant allelic missense mutations (Oily and Carboniferous) of Predicted gene 4951 (Gm4951) identified from a forward genetic screen in mice. GM4951 deficient mice developed NAFLD on high fat diet (HFD) with no changes in body weight or glucose metabolism. Moreover, HFD caused a reduction in the level of Gm4951, which in turn promoted the development of NAFLD. Predominantly expressed in hepatocytes, GM4951 was verified as an interferon inducible GTPase. The NAFLD in Gm4951 knockout mice was associated with decreased lipid oxidation in the liver and no defect in hepatic lipid secretion. After lipid loading, hepatocyte GM4951 translocated to lipid droplets (LDs), bringing with it hydroxysteroid 17β-dehydrogenase 13 (HSD17B13), which in the absence of GM4951 did not undergo this translocation. We identified a rare non-obese mouse model of NAFLD caused by GM4951 deficiency and define a critical role for GTPase-mediated translocation in hepatic lipid metabolism.
Project description:The protein interactome of the human C3orf33 protein was explored in U2OS cells through affinity purification-mass spectrometry (AP-MS). Specifically, U2OS cells stably expressing C3orf33 with either an N-terminus or a C-terminus 3XFlag tag were initially lysed using RIPA buffer. Subsequently, the proteins associated with C3orf33 were purified with anti-flag magnetic beads. Proteins were then eluted from the beads using competition with 3×Flag peptides and subsequently analyzed with mass spectrometry.
Project description:We report the results of ChIP-Seq experiment investigating the Rrf2 family transciptional regulator RsrR (putativeily name Redox sensitive response regulator). The experiment utilised an in trans copy of rsrR with an N-terminal 3xFlag tag encoded on the plasmid pMS82 (containing a phiBT1 integration site). The experiment utilised beads associated with anti-flag antibodies, specific for the Flag tagged protein and a WT host strain. Exo nuclease treatment was carried prior to immunoprecipitation to improve the resolution of the binding site. We report >600 target binding sites for the RsrR regulon encompasing a broad range of functional targets with specific refernce to those associate with NADPH and NADH metabolism and synthesis.
Project description:The aim of this analysis is to identify the difference between interaction partners of MRG15 wild type and Chromodomain and MRG domain mutants upon UV irradiation. Therefore, MRG15 wild type and mutants (N-terminal FLAG tag) were overexpressed in U2OS cells. After UV irradiation, immunoprecipitation of the constructs was performed using FLAG M2 affinity gel. The constructs were eluted from the beads with 3xFLAG peptide.
Project description:To investigate how α4 (IGBP1) regulates insulin signaling in adipocytes, we conducted analysis of proteins that interact with α4 by using brown preadipocyte cell lines (WT-1 cells) transfected with 3XFlag-tagged α4 protein, followed by immunoprecipitation of associated proteins using anti-Flag beads and mass spectroscopic proteomic analysis.
Project description:Putative subunits of the Mg-protoporphyrin monomethylester oxidative cyclase from Synechocystis PCC 6803, sll1214 and sll1874 were expressed with N-terminal 3xFLAG-tags in separate transformants.. Solubilised thylakoid membranes from these 2 transformants were incubated with anti-FLAG agarose and after extensive washing, the FLAG-tagged proteins, together with putative interaction partners were desorbed with FLAG peptide. The eluates were denatured, reduced, alkylated and digested with trypsin. The resulting peptides were cleaned by cation exchange and desalted, then analysed by nanoLC-MS/MS. The FLAG pulldown experiment was also carried out with a thylakoid membrane preparation from wild-type with no FLAG-tagged proteins as a control. Protein identification was by database searching with Mascot.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of JMJ28 in 14 days old arabidopsis. By obtaining sequence from chromatin immunoprecipitated DNA, we generated genome-wide JMJ28-binding maps of 14 days old arabidopsis. To reveal bound genes by JMJ28, chimeric protein JMJ28-3xFLAG was expressed under JMJ28 promoter in jmj28 (JMJ28pro:JMJ28:3xFLAG/ jmj28). ChIP was performed using anti-FLAG antibody (FLAG-M2, F1804; SIGMA), and ChIP DNA were analyzed by Illumina Novaseq 6000.