Project description:Monobromobimane (mBBr) labelling: mBBr labelling was carried out either immediately prior to 42°C, two hour heat shock, or immediately following heat shock. Medium was removed from culture flasks and cells were washed using PBS. Cells were then labelled by incubation at 37°C with 6 mL of 400 µM mBBr (Sigma-Aldrich, #B4380) for 10 minutes. Following labeling, 6 ml of 2 mM L-glutathione reduced (GSH, SigmaAldrich, #G4251) in PBS was added to quench the mBBr reaction. The quenched mBBr solution was removed and cells were washed with PBS. Mass spectrometry (MS) sample preparation, analysis and data processing: Six 1.6 mm steel beads (Next Advance Inc.) were added to the cell pellet tube with 30 µL SL-DOC (1.1% (w/w) sodium dodecyl sulfate (Sigma), 0.3% (w/w) sodium deoxycholate (Sigma), 25 mM ammonium bicarbonate (AB, Fluka), in de-ionised (DI) water containing 0.1% (v/v) protease inhibitor cocktail (Sigma), and 0.1% (v/v) phosphotase inhibitor cocktail (Sigma). Cells were homogen
Project description:ATAC-seq analysis was performed in a T-ALL cell line (HPB-ALL) which overexpresses TAL1 and LMO1 to analyze chromatin accessibility.
Project description:Enteric helminths form intimate physical connections with the intestinal epithelium, yet their ability to directly alter epithelial stem cell fate has not been resolved. Here we demonstrate that infection of mice with the symbiotic parasite Heligmosomoides polygyrus bakeri (Hpb), reprograms the intestinal epithelium into a fetal-like state marked by the emergence of Clusterin-expressing revival stem cells (revSCs). Organoid-based studies using parasite-derived excretory/secretory products reveal that Hpb-mediated revSC generation occurs independent of host-derived immune signals and inhibits type 2 cytokine-driven differentiation of secretory epithelial lineages that promote their expulsion. Reciprocally, type 2 cytokine signals limit revSC differentiation and, consequently, Hpb fitness indicating that helminths compete with their host for control of the intestinal stem cell niche to promote continuation of their life cycle.
Project description:Enteric helminths form intimate physical connections with the intestinal epithelium, yet their ability to directly alter epithelial stem cell fate has not been resolved. Here we demonstrate that infection of mice with the symbiotic parasite Heligmosomoides polygyrus bakeri (Hpb), reprograms the intestinal epithelium into a fetal-like state marked by the emergence of Clusterin-expressing revival stem cells (revSCs). Organoid-based studies using parasite-derived excretory/secretory products reveal that Hpb-mediated revSC generation occurs independent of host-derived immune signals and inhibits type 2 cytokine-driven differentiation of secretory epithelial lineages that promote their expulsion. Reciprocally, type 2 cytokine signals limit revSC differentiation and, consequently, Hpb fitness indicating that helminths compete with their host for control of the intestinal stem cell niche to promote continuation of their life cycle.
Project description:This project aims to identify the potential interacting proteins of lignin biosynthetic P450 enzymes by affinity purification and LC-MS/MS analysis. To this end, the genomic pieces of three lignin biosynthetic P450 genes C4H, C3’H and F5H were cloned into pMDCpC4H-HPB vector to generate HPB-fusion constructs driven by C4H promoter. The resulted constructs were then respectively transformed into ref3-3 (for C4H), Col-0 WT (for C3'H), and fah1-2 (for F5H) backgrounds and pMDCpC4H-HPB empty vector (as control) was transformed into Col-0 WT to generate transgenic plants. Arabidopsis T2 transgenic plants were grown for 5 weeks in soil and 15 grams of stem tissues were collected for protein extraction. Membrane protein complex was purified by affinity purification using streptavidin beads and subject to LC-MS/MS analysis. Two biological repeats were performed for each construct. For the first repeat (gel-base proteomics), each P450 construct has its control since each experiment was performed separately. For the second repeat (affinity purification,AP-MS), one control was shared for all the three P450 genes.
