Project description:We applied quantitative mass spectrometry (MS)-based proteomics to study the roles of Cbl and Cbl-b in long-term signaling responses related to neurite outgrowth and differentiation of SH-SY5Y neuroblastoma cells. Using stable isotope labeling by amino acids in cell culture (SILAC) and tandem mass tag (TMT)-labeling in combination with off-line high-pH reversed-phase fractionation and LC-MS/MS we analyzed how Cbl and Cbl-b depletion by siRNA affected the proteome, phosphoproteome and ubiquitylome of the neuroblastoma cells. SILAC proteome SILAC (Light Arg0/Lys0, medium Arg6/Lys4, heavy Arg10/Lys8) SH-SY5Y cells were treated with Cbl and Cbl-b or control (GFP) siRNA for 72 hours. For combined stimulation with ligand cocktail (FGF-2, IGF-1 PDGF-BB, TGFα) cells were treated with ligands for 48 h. Samples were analyzed in triplicates with set-up as described below: Set-up 1, 3 replicates (R1-3): Light: siGFP, Heavy: siCbl/siCbl-b Set-up 2, 3 replicates (E1-3): Light: siGFP + ligand cocktail, Medium: siCbl/siCbl-b, Heavy: siCbl/siCbl-b + ligand cocktail TMT phosphoproteome and proteome SH-SY5Y cells were treated with Cbl and Cbl-b siRNA, control (GFP) siRNA or Retinoic acid (RA) for 24 hours. Samples were prepared in triplicates and labelled with TMT10-plex reagents according to the set-up below: TMT10-126: siGFP E1 TMT10-127N: siCbl/siCbl-b E1 TMT10-127C: Retinoic acid E1 TMT10-128N: siGFP E2 TMT10-128C: siCbl/siCbl-b E2 TMT10-129N: Retinoic acid E2 TMT10-129C: siGFP E3 TMT10-130N: siCbl/siCbl-b E3 TMT10-130C: Retinoic acid E3 TMT10-131: Mix of the 9 samples SILAC Ubiquitin pulldown SILAC (Light Arg0/Lys0, heavy Arg10/Lys8) SH-SY5Y cells were treated with Cbl and Cbl-b or control (GFP) siRNA for 24 hours. Samples were analyzed in duplicates with set-up as described below: Light: siGFP, Heavy: siCbl/siCbl-b
Project description:We report the development of a new computational method to assess differences in cell-cell interactions between conditions through utilizing single-cell RNA sequencing data. The pipeline, known as Cell Interaction Network Inference from Single-cell Expression data (CINS), combines Bayesian network analysis with regression-based modeling to identify differential cell type interactions and the proteins that underlie these interactions.
Project description:We present LASSIM, which is a toolbox built to build and infer parameters within mechanistic models on a genomic scale. This is made possible due to a property shared across biological systems, namely the existence of a subset of master regulators, here denoted the core system. The introduction of a core system of genes simplifies the inference into small solvable sub-problems, and implies that all main regulatory actions on peripheral genes come from a small set of regulator genes. This separation allows substantial parts of computations to be solved in parallel, i.e. permitting the use of a computer cluster, which substantially reduces the time for the computation to finish.
Project description:We present LASSIM, which is a toolbox built to build and infer parameters within mechanistic models on a genomic scale. This is made possible due to a property shared across biological systems, namely the existence of a subset of master regulators, here denoted the core system. The introduction of a core system of genes simplifies the inference into small solvable sub-problems, and implies that all main regulatory actions on peripheral genes come from a small set of regulator genes. This separation allows substantial parts of computations to be solved in parallel, i.e. permitting the use of a computer cluster, which substantially reduces the time for the computation to finish.
Project description:We present LASSIM, which is a toolbox built to build and infer parameters within mechanistic models on a genomic scale. This is made possible due to a property shared across biological systems, namely the existence of a subset of master regulators, here denoted the core system. The introduction of a core system of genes simplifies the inference into small solvable sub-problems, and implies that all main regulatory actions on peripheral genes come from a small set of regulator genes. This separation allows substantial parts of computations to be solved in parallel, i.e. permitting the use of a computer cluster, which substantially reduces the time for the computation to finish.
Project description:We examine the role of Klf6 in oligodendrocyte progenitor cells and determine that Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), a key controller of nuclear trafficking in oligodendrocytes. Examination of expression profiles of 2 different cell stages exposed to siRNA vs. control
Project description:We found that USF2 is repressor of lysosomal genes. To find the interacting co-repressor complex of USF2, we performed USF2 complex purification and mass spectrography.