Project description:Pulse (P) - Chase (C) SILAC analysis of mitochondrial complex assembly
Bogenhagen et al (2018) Cell Reports
Pulse-chase times in hours (P#C#)
Project description:We developed a simple method that combined biochemical mitochondria isolation and pulse SILAC approach to monitor the mitochondrial translation. Our approach allows us to quantify 12 out of the 13 mitochondrial translation products, the highest coverage among analogous methods reported, and provide a global picture of (post-)translational regulation in mitochondria. Replicate 1: H-CRP_M-DMSO, Replicate 2: M-CRP_H-DMSO.
Project description:Pulse chase SILAC was used to identify protein turnover within human macrophages infected with mycobacterium tuberculosis CDC1551, a ppe38-71 mutant strain, a complemented strain and an uninfected control.
Project description:Manassantin A is a natural product that has been isolated from the perennial herb Saururus chinensis Baill and the aquatic plant Saururus cernuus. Manassantin A has been shown to possess potent hypoxia inducible factor 1 alpha (HIF-1α) inhibitory activity in a cell-based assay screen of thousands of natural products. Manassantin A holds promise as an anti-cancer drug since it has been shown to selectively target tumor cells over normal cells. Due to the complex biological pathways involved in cancer and hypoxia, it is difficult to determine the mode-of-action by which manassantin A inhibits HIF-1. While some of the biological activities of manassantin A have been discovered in various cell-based activity assays, the molecular basis of manassantin A’s biological activities is not well characterized. The proteins in a hypoxic MDA-MB-231 cell lysate were screened for interactions with manassantin A using large scale experiments to uncover novel manassantin A interactions that lead to the drug’s HIF-1 inhibition and anti-cancer activity. Two energetics-based approaches were utilized in this manassantin A mode-of-action study: iTRAQ-SPROX and SILAC-Pulse Proteolysis. In these energetics-based approaches, protein stability is measured using the chemical denaturant dependence of either a methionine oxidation reaction (iTRAQ-SPROX) or a thermolysin protease digest (SILAC-Pulse Proteolysis). Using boh of these techniques, the stability of proteins in the absence and presence of excess manassantin A was monitored to assess ligand-induced protein stability changes.
Project description:To measure translational efficiency in FMRP depletion, we purified RNAs from either wild-type or FMR1-knockout (FMR1-KO) SH-SY5Y cells generated for SILAC coupled to LC-MS/MS analysis and performed RNA-seq to quantitate mRNA abundance to normalize their protein abundance.
Project description:Pulse chase measurements using thiouracil (DTU) labeling via UPRT and chasing with uracil Data from tachyzoites is labeled "DTU Pulse Chase". Two independent pulse chase experiments were performed in tachyzoites, pulse chase 1 and 2. Duplicate arrays at each timepoint were performed for pulse chase 2 (2 a and b). Data from bradyzoites are labeled "DTU Bradyzoite Pulse Chase". Two independent pulse chase experiments were performed in bradyzoites and a single set of arrays were performed for each experiment. Just one chase timepoint was used in the bradyzoite experiments, the 2 hour chase. An RNA stablity experiment design type examines stability and/or decay of RNA transcripts. Keywords: RNA_stability_design
Project description:Cells were grown in light (R0K0) SILAC media (AthenaES) for 7 days, they were then incubated in light media for 24 hours in respective condition (normoxia neutral (NN) pH: 7.4; hypoxia neutral (HN)1% O2, pH: 7.4; hypoxia acidosis (HA), 1% O2, pH=6) and pulsed with heavy (R10K8) SILAC media (AthenaES) for 16 hr (TMT-pSILAC) following treatment.
Project description:Pulse chase measurements using thiouracil (DTU) labeling via UPRT and chasing with uracil Data from tachyzoites is labeled "DTU Pulse Chase". Two independent pulse chase experiments were performed in tachyzoites, pulse chase 1 and 2. Duplicate arrays at each timepoint were performed for pulse chase 2 (2 a and b). Data from bradyzoites are labeled "DTU Bradyzoite Pulse Chase". Two independent pulse chase experiments were performed in bradyzoites and a single set of arrays were performed for each experiment. Just one chase timepoint was used in the bradyzoite experiments, the 2 hour chase. An RNA stablity experiment design type examines stability and/or decay of RNA transcripts. User Defined