Project description:TAP-GluN1 (840 kDa and 1.5 MDa), PSD95-TAP (1.5 MDa) and WT (control; 1.5 MDa) native complexes from Grin1^TAP/TAP, Dlg4^TAP/TAP, and WT mouse forebrain (cortex and hippocampus), respectively. Purified samples were seperated by blue native PAGE. Bands were excised, digested with trypsin. Peptides were identified by LC-MS/MS.
Project description:MCF-7 TET Off cells (MCF-7 wt) were used to produce stable clones expressing ER-beta tagged with TAP-tag respectively at the C-term and at the N-term (C-TAP-ER-beta and N-TAP-ER-beta) or expressing ER-alpha tagged (C-TAP-ER-alpha) as previously described.Cells were cultured in standard growth conditions, then were lysed and RNA was extracted using TRIzol method.Total RNA were fluorescently labelled, amplified and hybridized in triplicate (MCF7-TAP and C-TAP-ER-alpha) and in duplicate (C-TAP-ER-beta_A, C-TAP-ER-beta_B, N-TAP-ER-beta) for 18 hours on Illumina v2 MicroRNA Expression BeadChips and after scanning, analysis was performed with GenomeStudio v.2010.1 software, for quality control and miRNA expression analysis.
Project description:MCF-7 TET Off cells (MCF-7 wt) were used to produce stable clones expressing ER-beta tagged with TAP-tag respectively at the C-term and at the N-term (C-TAP-ER-beta and N-TAP-ER-beta) or expressing ER-alpha tagged (C-TAP-ER-alpha). All were grown in Dulbecco's modified Eagle's medium (DMEM). Then cells were lysed and RNA extracted were pooled. For mRNA expression profiling, 500 ng total RNA were reverse transcribed and used for synthesis of cDNA and biotinylated cRNA. Finally cRNA were hybridized for 18 hours on Illumina HumanHT-12 v3.0 BeadChips and after scanning, data analysis was performed.
Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein We purified TAP::ALG-1 complexes from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2, serving as a mock control) animals and hybridized the associated mRNAs to two-color microarrays RNA isolated from WS4303 and N2 (mock control) animals was analyzed for each IP sample and for each total worm extract sample. Three independent biological replicates have been performed. Long-oligo whole-genome C. elegans arrays, produced by the Genome Sequencing Center at Washington University in St. Louis (http://genome.wustl.edu/genome/celegans/microarray/ma_gen_info.cgi), were used for analysis. Log2 ratios (immunopurified RNA over total RNA from extract) were calculated for each gene. Microarrays generated from WS4303 animals were compared to microarrays generated from N2 mock control animals by a Student’s two sample t test.
Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as wild typeand the transgenic WS5041 animals as mir-58. We compared the mRNA population that coimmunopurified with TAP::ALG-1 from synchronized L4 stage wild-type animals with that from synchronized L4 stage mir-58 mutant animals by one-color Affymetrix gene arrays. miR-58 target mRNAs should be specifically underrepresented in the latter samples. Strains WS4303 (wt) and WS5041 (mir-58) were used for TAP::ALG-1 IPs. All experiments were conducted in three independent replicates. For each replicate, WS4303 and WS5041 were grown in parallel. 150 ng of TAP::ALG-1 associated RNA isolated from synchronized late L4 animals were sent to the GeneCore facilty in Heidelberg, Germany (http://www.genecore.embl.de/index.cfm), and the microarray data were generated according to their standard protocol (Weinmann et al. 2009).
Project description:We used DNA microarrays to define the physiological roles of the Tap efflux pump in M. bovis BCG during the exponential and the stationary phase of in vitro growth. For this purpose we constructed a M. bovis BCG strain in which the tap gene was inactivated by the insertion of a hygromycin resistance cassette (?-hyg). When the gene expression patterns of the tap mutant were compared to the wild-type strain, almost no differences were observed during exponential growth; only seven genes slightly increased their expression. In contrast, more that 100 genes showed a variation in their level of expression during stationary growth. More than ten representative genes were chosen from the microarray experiments and their expression was measured by quantitative RT-PCR using sigA as invariant internal control. In support to the gene expression profiling data, the mRNA levels of all selected genes was significantly different in the tap mutant strain relative to control. A functional category analysis (http://tuberculist.epfl.ch/index.html) of the genes differentially expressed revealed a high proportion belonging to the Virulence, Detoxification, Adaptation (VDA), Intermediary Metabolism and Respiration (IMR), Conserved Hypotheticals (CH), and Cell Wall and Cell Processing (CWCP) categories suggesting a major adaptation to a stress generated by inactivation of the tap efflux pump gene. We compared the global gene expression of the tap mutant versus the wild-type strain of M. bovis BCG during the exponential (one week; OD540= 0.2-0.3) and stationary (six weeks; OD540= 0.8-1.0) growth. Hybridizations were performed using RNA extracted from two different biological samples. Each sample was hybridized twice through swap labeling of the respective cDNAs.