Project description:The pentameric glycine receptor (GlyR), comprising the α1 and β subunits, is a major inhibitory ionotropic receptor in brainstem and spinal cord. GlyRs interact with gephyrin (GPHN), a scaffold protein that anchors the GlyR in the plasma membrane and enables it to form clusters in glycinergic postsynapses. Using an interaction proteomics approach, we provides evidence of the ArfGEFs IQ motif and Sec7 domain 3 (IQSEC3) and IQ motif and Sec7 domain 2 (IQSEC2) as two novel synaptic proteins interacting with GlyR complexes. When the affinity-isolated GlyR complexes were fractionated by blue native gel electrophoresis and characterized by mass spectrometry, GlyR α1β-GPHN appeared as the most abundant complex with a molecular weight of approximately 1 MDa, and GlyR α1β-GPHN-IQSEC3 as a minor protein complex of approximately 1.2 MDa. A third GlyR α1β-GPHN-IQSEC2 complex existed at the lowest amount with a mass similar to the IQSEC3-containing complex. Using yeast two-hybrid we demonstrate that IQSEC3 interacts with the GlyR complex by binding to the GPHN G domain at the N-terminal of the IQSEC3 IQ-like domain. Our data provide direct evidence of the interaction of IQSEC3 with GlyR-GPHN complexes, underscoring a potential role of these ArfGEFs in the function of glycinergic synapses.
Project description:EndoMAP aims to systematically dissect the human endosomal protein complexes. To purify endosomes from human cells, a modified Endo-IP protocol was developed to be compatible with complexomics methodologies and evaluated by DIA-MS. Three independent biological replicates of human endosomes purified by the modified Endo-IP protocol were subjected to Blue Native PolyAcrylamide Gel Electrophoresis (BN-PAGE) co-fractionation to identify possible protein complexes based on correlation profiling.
Project description:We identified 737 differentially expressed genes, including 430 upregulated genes and 307 downregulated genes, by calculating the gene FPKM in each sample and conducting differential gene analysis. Gene ontology analysis and KEGG pathway enrichment analysis suggested that blue light influenced visual perception, sensory perception of light stimulus, phototransduction, and JAK-STAT signaling pathways. Differential lncRNA, circRNA and miRNA analysis suggested that blue light exposure affected pathways for retinal cone cell development and phototransduction, among others.
Project description:The discovery of Toll-like receptors (TLRs) represented a significant breakthrough that paved the way for the study of host-pathogen interactions in innate immunity. However, there are still major gaps in understanding TLR function, especially the early dynamics of downstream TLR pathways remains less clear. We characterize a label-free optical biosensor-based assay as a powerful method to detect TLR activation in a native and label-free environment and to delineate the dynamics of TLR pathway activation. To gain a deeper insight into the biological processes underlying the ligand-specific signal traces of different LPS chemotypes in various cell types, RNA-seq analysis of transcriptional changes in HEK Blue hTLR4 reporter cells was performed. After 3 hours of stimulation with LPS E. coli or LPS S. minnesota, HEK Blue hTLR4 reporter cells showed significant changes in RNA expression compared to the unstimulated control.
Project description:A tuberization inhibitor has long been postulated, but not yet found. We found that blue light inhibits tuberization in Norland, a day-neutral variety of Solanum tuberosum L. Tissue-cultured plants formed tubers within 8 weeks under continuous darkness, and white, red, or far-red light. Preliminary experiments indicated that a one- or two-day exposure to blue light after 3-4 weeks of dark treatment will inhibit tuber formation in ‘Norland’ plants. Using this system and expression profiling, we may be able to identify candidate tuberization inhibitors. 'Norland' plants (subcultured from existing cultures and grown for two weeks under continuous 100 umol/m2-s white fluorescent light) were placed in tuber-inducing media containing 6% sucrose, vitamins, MS salts, and kinetin (2.5 mg/L). Tubes containing plants were wrapped in two layers of aluminum foil. After 3 weeks and 2 days, half of the tubes were exposed to 6-7 umol/m2-s blue light. The other half of the tubes were left in darkness (controls). After 2 days, all plants were harvested and frozen in liquid nitrogen. Plants exposed to blue light were harvested under blue light. Control plants were harvested under < 2 umol/m2-s light conditions. All plant transfers were done at 1700 (5 PM) to avoid possible complications due to circadian effects. Experiments were performed four times, from subculture to harvest. RNA was extracted from stem and leaf tissue of plants using the Qiagen RNeasy Plant Mini kit. Extracted RNA was then converted to dsDNA using the Invitrogen protocol and reagents for double stranded cDNA synthesis. The resulting dsDNA was in vitro transcribed into amplified RNA using the Ambion procedure and reagents for in vitro transcription. cDNA was purified using Qiagen MinElute columns and protocol. Amplified RNA was purified using Ambion columns or Qiagen RNeasy columns and the Ambion protocol, and quantified using RiboGreen dye fluorometry. Keywords: Direct comparison
Project description:Blue-native gel electrophoresis (BN) is a powerful method for protein separation. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) it enables large-scale identification of protein complexes and their subunits. Current BN-MS approaches, however, are limited in size resolution, comprehensiveness and quantification. Here, we present a new methodology combining defined sub-mm slicing of BN gels by a cryo-microtome with high-performance LC-MS/MS and label-free quantification of proteins. Application of this cryo-slicing BN-MS approach (csBN-MS) to mitochondria from brain demonstrated a high degree of comprehensiveness, quantification accuracy and size resolution. The technique provided abundance-mass profiles for 743 mitochondrial proteins including all canonical subunits of the oxidative respiratory chain assembled into 13 distinct (super)complexes. Moreover, the data revealed COX7R as a constitutive subunit of distinct supercomplexes and identified novel assemblies of porins and TOM proteins. Together, csBN-MS enables quantitative profiling of complexomes with resolution close to the limits of native gel electrophoresis.
Project description:Analysis of the subunits composition of the thylakoids protein complexes in Picea abies (Norway spruce) by means of two-dimensional large-pore Blue-Native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D lpBN/SDS-PAGE) and in-gel tryptic digestion of single spots.