Project description:Identification of biotinylated proteins through pull-down. RAW264.7 cells stably expressing a Dox-inducible TurboID-TTP fusion protein were treated with LPS or Epoxomicin for 4 h, followed by 15 min of biotin labeling in the cell medium. TurboID without fusion was used as a control.
Project description:Identifying interactors of IRF1 and STAT1 in bone marrow-derived macrophages during type I and type II interferon treatment using the proximity-dependent labeling approach TurboID followed by Mass Spectrometry
Project description:The analysis of differential interactome of Pol II pS5 in DIDO WT, KO and ΔSPOC HEK293T cells using Pol II pS5 (3E8) antibody coupled to Protein G beads (no crosslinking).
Project description:In this set of proof of principle experiments we compare the proximity interactomes obtained for two C. elegans centrosomal proteins, SPD-5 and PLK-1, by direct TurboID and indirect, GFP nanobody-targeted, TurboID in a range of different tissues, embryos, germ cell precursors and ciliated neurons.
Project description:Goal was to identify N-recognins for Arginine and Leucine by comparison of biotinylated proteins from 1 week old whole Arabidopsis seedlings constitutively expressing the bait constructs R-degron-Turbo or L-degron-Turbo. M-degron-Turbo served as a negative control. The plants were treated with 50µM biotin and either the proteasome inhibitor Bortezomib (10µM) or the inhibitor of vacuolar acidification Concanamycin A (1µM) or DMSO one hour before harvest.
Project description:Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARFBP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells. MIZ1 and MYC ChIPseq experiments in HUWE1 inhibitor-treated Ls174T cells as well as RNAseq experiments in HUWE1- or MIZ1-depleted Ls174T cells after HUWE1 inhibitor treatment. Sequencing was performed on an Illumina Genome Analyzer IIx.
Project description:Selective autophagy contributes to the removal of harmful proteins from the cytoplasm. This cargo material is selected by cargo receptors such as p62/SQSTM1 and finally degraded. However, the molecular composition of the p62 condensates and therefore the nature of the cargo delivered by them is incompletely understood. In order to obtain insights into their composition, we have developed a method to isolate these condensates followed by TMT and Label Free mass spectrometry-based identification of their protein content.
Project description:Chemotherapy-induced peripheral neuropathy (CIPN) is a serious side effect of cancer treatment, often leading to cessation of therapy due to lack of adequate treatment options. In the peripheral nervous system the chemotherapeutic agent paclitaxel impairs mitochondrial function and induces vast changes in gene expression. However, changes at the protein level remain so far unexplored. Here we used an unbiased approach of quantitative proteome profiling in a clinically-relevant rat model of paclitaxel-induced peripheral neuropathy. We analysed two critical timepoints: (1) day 7, equivalent to 1 day after cessation of paclitaxel treatment, prior to neuropathy development; (2) approximately 4 weeks after paclitaxel initiation, when neuropathy has developed.
Project description:To monitor selective degradation of ClpC1 and ClpC2 using BacPROTAC compounds in M. smegmatis we performed a LFQ proteomics analysis treating the cells at bactericidal compound concentration. We detected degradation of both proteins and additional dysregulation of the mycobacterial PQC system.