ABSTRACT: Huh7 cells with expression of Flag CLOCK were treated with IGF1. An immunoprecipitation assay was performed , and immunoprecipitants were subjected to masspec analysis.
INSTRUMENT(S): Orbitrap Elite mass spectrometer(Thermo)
Project description:Huh7 cells with expression of Flag-GPX4 were treated with IGF1. An immunoprecipitation assay was performed, and immunoprecipitates were subjected to masspec analysis.
Project description:The timing of many molecular and physiological processes in plants occurs at a specific time of day. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT INDUCIBLE AND CLOCK REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover new connections within the clock and between other pathways, here we use affinity purification coupled with mass spectrometry to discover time-of-day-specific protein interactors of the RVE8-LNK1/2 complex. Our clock proteins are tagged with a 6X-His-3X-FLAG C-terminal (HFC) affinity tag and purified through anti-FLAG immunoprecipitation and His-tag isolation affinity purification. We also performed affinity purification-mass spectrometry of 35S::YFP-COR27 and 35S::GFP-COR28, which were immunoprecipitated with anti-GFP TRAP beads. Experiment was performed on 10-day-old seedlings grown under 12 hours light: 12 hours dark 22 degrees Celsius.
Project description:A375 cells transduced with empty pLVX or pLVX-SPANXA-FLAG were subjected to immunoprecipitation using Flag beads. Elution from beads was performed by incubation with 3XFlag peptide for 1h at 4 C and eluates were subjected to tryptic digestion followed by LC-MS/MS, as described previously (PMID: 30992132). Raw data were analyzed using MaxQuant (v1.6.0.1) with default settings.
Project description:The molecular mechanisms underlying human brain evolution are not fully understood; however, previous work suggested that expression of the transcription factor CLOCK in the human cortex might be relevant to human cognition and disease. In this study, we investigated this novel transcriptional role for CLOCK in human neurons by performing chromatin-immunoprecipitation sequencing for endogenous CLOCK in adult neocortex and RNA-sequencing following CLOCK knockdown in differentiated human neurons in vitro. These data suggested that CLOCK regulates expression of genes involved in neuronal migration, and a functional assay showed that CLOCK knockdown increased neuronal migratory distance. Furthermore, dysregulation of CLOCK disrupts co-expressed networks of genes implicated in neuropsychiatric disorders, and the expression of these networks are driven by hub genes with human-specific patterns of expression. Thus, these data support a role for CLOCK-regulated transcriptional cascades involved in human brain evolution and function.
Project description:Circadian rhythms are a series of endogenous autonomous 24-hour oscillations generated by the circadian clock. At the molecular level, the circadian clock is generated by a transcription-translation feedback loop, where BMAL1 and CLOCK transcription factors of the positive arm activate the expression of CRYPTOCHROME and PERIOD (PER) genes of the negative arm as well as the circadian clock-regulated genes. In this project, we aimed at finding the interactome of PER2 protein in human U2OS osteosarcoma cell line using proximity-dependent biotin identification (BioID) technique. U2OS clones overexpressing PER2-BioID2 or BioID2 were treated with dexamethasone in order to reset the circadian rhythm, and cells were then incubated in biotin-containing media for 12 hours to label the proteins in close proximity of PER2-BioID2. Samples were collected after 36 and 48 hours of the resetting to identify the labeled proteins by mass spectrometry. In addition to known interactors such as CRY1 and CRY2, many novel interactors were identified. In summary, we obtained a network of PER2 interactome and confirmed some of the novel interactions using classical the co-immunoprecipitation method.
Project description:To identify the substrates of METTL5, we used crosslinking-assisted immunoprecipitation of overexpressed, FLAG-METTL5 followed by high throughput sequencing.
Project description:We show that the cyclin-dependent kinase 5 (CDK5) regulates the mammalian circadian clock via phosphorylation of PER2. CDK5 phosphorylated PER2 at serine residue 394 (S394) as shown by an in vitro kinase assay.
Project description:Endogenous ZFC3H1 was tagged in 293T cells. Dignam cellular extracts where subjected to sequential FLAG and HA immunoprecipitation and elution. Complexes were then analyzed using mass spectrometry at the taplin mass spectrometry facility. The 2 samples are FLA-HA IP in wild-type 293T cells and Flag-HA IP in FH-ZFC3H1 293T cells.