Pulse-chase SILAC analysis of mitochondrial complex assembly
Ontology highlight
ABSTRACT: 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: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: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: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
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.
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:Thalidomide and its derivatives lenalidomide and pomalidomide (IMiDs) are effective treatments of hematologic malignancies. It was shown that IMiDs impart gain of function properties to the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) ubiquitin ligase that enable binding, ubiquitination and degradation of key therapeutic targets such as IKFZ1, IKZF3 and CSNK1A1. While these substrates have been implicated as efficacy targets in multiple myeloma (MM) and 5q deletion associated myelodysplastic syndrome (del(5q)-MDS), other targets likely exist. Using a pulse-chase SILAC mass spectrometry-based proteomics approach, we demonstrate that lenalidomide induces the ubiquitination and degradation of ZFP91. We establish that ZFP91 is a bona fide IMiD dependent CRL4CRBN substrate and further show that ZFP91 harbors a zinc finger (ZnF) motif, related to the IKZF1/3 ZnF, critical for IMiD dependent CRBN binding. These findings demonstrate that single time point pulse-chase SILAC mass spectrometry-based proteomics (pSILAC-MS) is a sensitive approach for target identification of small molecules inducing selective protein degradation.
Project description:Nascent RNA was tagged with EU, via EC feeding, in targeted cells (neuroblasts or neurons) then purified after the initial pulse labeling or after a chase using media containing excess unmodified uridine
Project description:We developed a pulse-chase method in where fully SILAC (heavy, medium-heavy and Light) labelled cells were pulsed with Azidohomoalanine (AHA) and then chased for different length of times. These experiments were performed with 0, 1, 2, 4, 8, 16 and 13 h of chase. Data from 3 biological replicates are provided. Each replicate contains data from 3 experiments (0, 1, 2 and 0, 4, 8 and 0, 16, 32 hours chase) in where the 0 h time point is always heavy labelled followed by the medium and light labelled time points. In addition, AHA p-c experiments were performed using only 0, 4 and 8 h chase times in combination with different inhibitor combinations (MG132, Actinomycin D and Wortmanninin + Bafilomycin A1) or control (DMSO). Also, a control enrichment data set was created. Here heavy SILAC labelled cells were pulsed with AHA before being mixed with light cells that were not pulsed with AHA. This was followed by the click reaction and enrichment of AHA containing proteins. Label-swap experiments were also performed. Finally, a SILAC p-c data set is provided. Here light cells were pulsed with heavy (Arg10, Lys8) amino acids and then split in two. One half of the cells was chased in medium (Arg6, Lys4) amino acids and the other part was directly frozen. Label-swap experiments and experiments using different pulse and chase times were also performed. All provided datasets are from mouse fibroblast cells (NIH 3T3).