Armeniaspirol mechanism of action proteomics data set
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion ETD
ORGANISM(S): Bacillus Subtilis Subsp. Subtilis Str. 168 (ncbitaxon:224308)
SUBMITTER: 
Christopher Boddy
PROVIDER: MSV000087632 | MassIVE | Tue Jun 15 12:16:00 BST 2021
REPOSITORIES: MassIVE
Similar Datasets
Project description:Flag-YBX1 overexpressed T24 cells pellets were resuspended with 2 volume of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/ml RNase inhibitor), and incubated at 4 °C for 30 min with rotation. Then the lysate was centrifuged at 15 000 g for 20 min. Before incubating the lysate with Flag beads, 100ul were taken as input. The anti-Flag M2 magnetic beads (Sigma, 10 μl per mg lysate) were washed with NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/ml RNase inhibitor) four times. Cell lysate was mixed with M2 beads and incubated at 4 °C for 4 h with rotation. The beads were washed two times with 1 ml ice-cold NT2 buffer. Then the beads were subject to Micrococal nuclease (NEB) digestion (1:1 000 000 dilution) for 8 min at 37 °C. The beads were cooled on ice immediately for 5 min and washed two times with 1 ml ice-cold 1× PNK+EGTA buffer (50 mM Tris-HCl pH 7.5, 20 mM EDTA, 0.05% NP-40, 200 U/ml RNase inhibitor) and two times with 1 ml ice-cold 1× PK buffer (50 mM NaCl, 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.2% SDS, 200 U/ml RNase inhibitor). Then the beads were digested with 200 μl pre-heated (20 min at 50 °C) Proteinase K and RNAs were extracted with an equal volume of Acid-Phenol: Chloroform, pH 4.5 (Ambion). The RNAs were subjected to rRNA removal and Bisseq. The libraries were sequenced on the Illumina HiSeq X-Ten platform at Novogene (Tianjin, CA) with paired-end 150 bp read length.The m5C sites were called using meRanCall from meRanTK (FDR < 0.01).
2019-07-02 | GSE133625 | GEO
Project description:Immunoaffinity Purifications of Human Argonaute Twelve hours after transfections with mock or with miR-124, we washed each 15-cm plate once with phosphate-buffered saline (usually two plates were used per IP), then added 1 ml of 4 C lysis buffer (150 mM KCl, 25 mM Tris-HCl [pH 7.4], 5 mM Na-EDTA [pH 8.0], 0.5% Nonidet P-40, 0.5 mM DTT, 10 ul protease inhibitor cocktail [Pierce Cat# 78437], 100 U/ml SUPERaseIn [Ambion Cat# AM2694]). Following a 30-min incubation at 4 C, we scraped the plates, combined the lysates, and then spun them at 4 C for 30 min at 14,000 RPM in a microcentrifuge. We collected the supernatant and filtered it through a 0.45-um syringe filter. We froze an aliquot of lysate in liquid nitrogen for reference RNA isolation. We then added 0.22 mg/ml heparin to the lysate. We mixed the lysate with 2.5 mg of Dynal m-280 streptavidin beads (250 ul from original storage solution) coupled to biotinylated 4F9 ago antibody (~12.5 ug), which we equilibrated immediately prior to use by washing twice with 1 ml of lysis buffer. We incubated the beads with the lysate for 2 h at 4 C and then washed the beads twice with 1.25 ml of ice-cold lysis buffer for 5-min each. Five percent of the beads were frozen for SDS PAGE analysis after the second wash. RNA was extracted directly from the remaining beads using lysis buffer from Invitrogen's Micro-to-Midi kit (Invitrogen Cat# 12183-018). We purified RNA from the lysate and RNA extracted from the beads with the Micro-to-Midi kit as per vender's instructions, except that the percentage isopropanol used for binding to the column was 70%, instead of 33%, to promote the binding of small RNAs. RNA was amplified with ambion kit 1755. RNA from total cell lysate was labeled with cy3 and IPd RNA was labeled with cy5
2009-11-10 | GSE18834 | GEO
Project description:Analysis of the transcriptome of THP-1 cells upon Huh7 cell-derived ectosomes incubation. Monocytic THP-1 cells were incubated with or without ectosomes (10 mg/ml) derived from scramble sequence transfection (Scramble Ecto) or PKM2 knocking down (PKM2 KD Ecto) Huh7 cells for 24 h. Results provide a novel insight into monocyte differentiation.
2022-06-08 | GSE132435 | GEO
Project description:Colorectal cancer tissue sections were obtained according to the inclusion criteria. The formalin was used to immersed all cancer specimens. And tissues were cut to 5 μm thickness and placed on glass slides before staining. Endogenous peroxidase activity was inhibited and blocked by de-paraffinizing, rehydrating, and using 5% bovine serum albumin at 37ºC for 30 min. The treated sections were incubated with anti-FOS (promab 30360) at 4ºC overnight and washed three times with PBS. After that, it is required that incubation with secondary anti-peroxidation sunflower at 37ºC for 30 minutes. After washing three times again with PBS, the sections were developed in diaminobenzidine and microscopic images were made by light microscopy.
| 6033 | ecrin-mdr-crc
Project description:The chromatin fraction was isolated from 5x107 HeLa cells. Chromatin was digested in 100 mL of MNase (40 units/ mL ) reaction buffer for 3 min at 37 °C in a thermomixer (1,400 rpm). After addition of 10 L EGTA (25mM) to inactivate MNase, soluble digested chromatin was collected by 13,000 rpm centrifuge for 5 min. 10 mg of Pol II antibody was conjugated to magnetic protein A beads. The supernatant was diluted with 400 mL of NET-2 buffer and pSer2-Pol II antibody-conjugated beads were added. Incubated the IP reaction at 4 °C for 1 hr. The beads were washed with 1 mL of NET-2 buffer six times and 100 mL of 1xPNKT (1xPNK buffer and 0.05% Triton X-100) buffer once. Washed beads were incubated in 50 mL PNK reaction mix (1xPNKT, 1 mM ATP and 0.05 U/ml T4 PNK (NEB)) in Thermomixer at 37 °C, 1,400 rpm for 6 min. Beads were washed with 1 mL of NET-2 buffer once and RNA was extracted with Trizol reagent. RNA was suspended in urea Dye (7M Urea, 1xTBE, 0.1% BPB and 0.1% XC) and resolved on 6% TBU gel (Invitrogen) at 200 V for 5 min. Gels with 30-160 nt RNAs were collected. 0.2 mL tube was prepared with holes made with 25G needle and placed in a 1.5 mL tube. Gel fragments were placed in the layered tube and broken down by centrifugation at 12,000 rpm for 1 min. The small RNAs were eluted from gel using RNA elution buffer (1 M NaOAc and 1 mM EDTA) at 25 °C for 1 hr in Thermomixer (900 rpm). Eluted RNA was purified with SpinX column (Coster) with 2 glass filters (Millipore) and the flow-through RNA was ethanol precipitated.
2019-10-15 | GSE134102 | GEO
Project description:Parasite lysates were centrifuged (20,000 g, 4°C, 15 min). Beads (blank and with linker attached) were washed 3× with water then twice with lysis buffer. The lysate (~2 mg total protein) was first incubated with 2 mg blank beads for 30 min at 4°C with rotating agitation. The lysate was divided into two then incubated with either 1% DMSO or 100 µM DDD01510706 (competitor) for 30 min at 4°C with agitation. Finally, lysates were incubated with 2 mg of compound-bound beads for 1 h at 4°C with agitation. The beads were then washed 3× with wash buffer (0.8% (w/v) octyl β-D-glucopyranoside, 50 mM Tris pH 8.0, 5 mM EDTA, 1 mg/mL BSA) and 2× Tris-buffered saline (TBS; 50 mM Tris-Cl pH 7.5, 150 mM NaCl). Samples were run 1.5 cm into a Bis-Tris 10% (w/v) acrylamide gel and stained with Coomassie quick reagent for 30 min. The entire gel bands were removed and subjected to in-gel reduction with 10 mM dithiothreitol, alkylation with 50 mM iodoacetamide and digestion with 12.5 μg/mL trypsin (Pierce) for >16 h at 37°C. Recovered tryptic peptides were then vacuum dried prior to analysis
2022-10-15 | PXD033740 | Pride
Project description:Immunoaffinity Purifications of Human Argonaute Twelve hours after transfections with mock or with miR-124, we washed each 15-cm plate once with phosphate-buffered saline (usually two plates were used per IP), then added 1 ml of 4 C lysis buffer (150 mM KCl, 25 mM Tris-HCl [pH 7.4], 5 mM Na-EDTA [pH 8.0], 0.5% Nonidet P-40, 0.5 mM DTT, 10 ul protease inhibitor cocktail [Pierce Cat# 78437], 100 U/ml SUPERaseIn [Ambion Cat# AM2694]). Following a 30-min incubation at 4 C, we scraped the plates, combined the lysates, and then spun them at 4 C for 30 min at 14,000 RPM in a microcentrifuge. We collected the supernatant and filtered it through a 0.45-um syringe filter. We froze an aliquot of lysate in liquid nitrogen for reference RNA isolation. We then added 0.22 mg/ml heparin to the lysate. We mixed the lysate with 2.5 mg of Dynal m-280 streptavidin beads (250 ul from original storage solution) coupled to biotinylated 4F9 ago antibody (~12.5 ug), which we equilibrated immediately prior to use by washing twice with 1 ml of lysis buffer. We incubated the beads with the lysate for 2 h at 4 C and then washed the beads twice with 1.25 ml of ice-cold lysis buffer for 5-min each. Five percent of the beads were frozen for SDS PAGE analysis after the second wash. RNA was extracted directly from the remaining beads using lysis buffer from Invitrogen's Micro-to-Midi kit (Invitrogen Cat# 12183-018). We purified RNA from the lysate and RNA extracted from the beads with the Micro-to-Midi kit as per vender's instructions, except that the percentage isopropanol used for binding to the column was 70%, instead of 33%, to promote the binding of small RNAs. RNA was amplified with ambion kit 1755. RNA from total cell lysate was labeled with cy3 and IPd RNA was labeled with cy5 An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
2010-06-20 | E-GEOD-18834 | biostudies-arrayexpress
Project description:We investigated an in vitro experimental exposure model of the MutaMouse flat epithelial (FE1) cells exposed to a complex mixture of carcinogenic PAHs known as coal tar (SRM 1597a) using an Agilent 22k, oligo mouse array platform. Keywords: Toxicology, biomarkers discovery, stress response, complex mixture of carcinogens, PAHs The FE1 cells were exposed to two different sub-toxic concentrations of coal tar (SRM 1597a): 1 mg/ml and 4 mg/ml in a serum free media. After 6h exposure, the cells were washed twice with PBS and re-incubated in fresh media without coal tar. After 4h and 8h incubation, the media were collected for proteomic experiment and the cells were collected from the petri dishes for RNA extraction.
2010-12-09 | E-GEOD-19437 | biostudies-arrayexpress
Project description:Trophoblast stem (TS) cells are in vitro equivalents to the precursor cells of the placenta. In mice, TS cells can be derived either from polar trophectoderm (TE) of blastocyst outgrowths or from postimplantation extraembryonic ectoderm (ExE), originating from polar TE. Since their first successful derivation in 1998 TS cells have been cultured in serum-rich medium in the presence of fibroblast growth factor 4 (FGF4), the cofactor heparin and embryonic fibroblast conditioned medium. Here, we developed and tested a simple medium consisting of ten chemically defined ingredients for routine culture of TS cells. The defined medium (TX medium) supports growth and self-renewal of TS cells grown on Matrigel-coated dishes over multiple (>20) passages. TS cells cultured under these conditions retain expression of key trophoblast stem cell-associated markers. Global gene expression profiling of cells cultured in standard and TX media conditions revealed that 99.6 % of genes are similarly expressed. DNA methylation profiling confirmed the faithful propagation of epigenomic characteristics of TS cells cultured in TX compared to standard media. Importantly, TX media also supported the derivation of new TS cell lines directly from post implantation embryos. In addition, TS cells cultured in TX retain their ability to differentiate into all derivatives of the trophectodermal lineage in vitro and give rise to haemorrhagic lesions in nude mice, indistinguishable from cells grown in standard conditions. Further, injection into blastocysts proofs chimerization potential after TX culture. The fact that TX media formulation no longer requires fetal bovine serum and conditioned medium facilitates and standardizes the culture of this extraembryonic lineage in vitro. Cell culture. TS cell lines TS-LacZ and TS-L5 have been derived from blastocysts and post implantation embryos us according to standard protocols. Cells were grown on tissue culture plastic (TPP) at 37°C in humidified incubators containing 5% CO2 (Heraeus). For conventional stem cell culture cells were cultured in serum containing medium supplemented with 70% CM, 25 ng/ml human recombinant FGF4 (Reliatech) and 1 µg/ml Heparin (Sigma-Aldrich), hereafter named TS medium. For differentiation experiments TS medium without CM, FGF4 and heparin (TS -CMF4H) was used. For stem cell culture in defined medium cells were cultured on Matrigel-coated dishes in TX medium. TX medium formulation: DMEM/F12 (Life Technologies), 64 mg/l l-ascorbic acid-2-phosphate magnesium (Sigma), 14 microg/l sodium selenite (Sigma-Aldrich), 25 ng/ml human recombinant FGF4 (Reliatech), 19.4 mg/l insulin (Sigma-Aldrich), 543 mg/l NaHCO3 (Sigma-Aldrich), 10.7 mg/l holo-transferrin (Sigma-Aldrich), 2 ng/ml human recombinant TGF-beta1 (Peprotech), 1 microg/ml Heparin (Sigma-Aldrich), 2 mM L-glutamine (PAN-biotech), 1% penicillin and streptomycin (PAN-biotech). Medium was prepared without growth factors, stored at 4°C and used within two weeks. FGF4, heparin and TGFß were added to the medium prior to use. Medium was changed every other day. Cells were passaged when sub confluent, by incubation in trypsin-EDTA (PAN-biotech) for 4 min at 37°C. The enzyme was inactivated by addition of trypsin inhibitor (Life Technologies) in defined culture and by serum containing medium in case of conventional culture. Cells were dissociated and plated in fresh medium. Passage numbers are represented as x + y, where x represents the passage number at which cells were transferred to TX medium, and y being the passage number in TX. For cryopreservation, cells were harvested and pellets were re-suspended either in FBS/10%DMSO (standard conditions) or in KnockOut Serum Replacement/10%DMSO (Life Technologies). Plate coating. Tissue culture dishes for culture of TS cells in TX medium were coated with growth factor reduced Matrigel (BD Biosciences), which was diluted 1:30 in ice cold DMEM/F12 and incubated over night at 4°C. Coating solution was aspirated immediately before plating cells. For initial coating tests, plates were incubated either with Matrigel, poly-L-lysine, gelatine or fibronectin, respectively. Poly-L-lysine was used at 0.01 % (w/v) in PBS (PAN-biotech) for 30 min at 37°C and washed once with PBS prior to use. Gelatine coating was performed with 0.1% (w/v) gelatine solution in PBS for 30 min at 37°C. Dishes were incubated with fibronectin solution (16.7 µg/ml) for 30 min in the incubator, washed once with PBS and used thereafter. Isolation of TS cells from post-implantation (E6.5) embryos Embryos were dissected in PBS on day E6.5 and transferred in PBS/10% (v/v) FBS and kept on ice. Before dissociation, embryos were washed in PBS twice. Whole conceptuses were transferred into an U-bottom 96-well plate and incubated in 40 µl trypsin-EDTA (PAN-biotech) for 15 min in the incubator. The content of the well was pipetted up and down several times and transferred to a 24 well plate on pre-plated irradiated MEFS (gammaMEFs) in either TS medium with 1.5x FGF4 and heparin concentration (TS+1.5xF4H) or TX medium with 1.5x FGF4 and heparin (TX+1.5xF4H). TS cell colonies appeared after 2-4 days. Immunofluorescent staining, western blotting. Before staining, cells were washed with PBS once and fixed for 10 min with 4% paraformaldehyde (Sigma-Aldrich). Permeabilization of the cells was performed with 0.5 % (v/v) Triton X-100 (Sigma-Aldrich) in PBS for 10 min and subsequent incubation for 30 min in blocking buffer (2% bovine serum albumin [Sigma-Aldrich], 0.1% (v/v) Triton X-100 in PBS). Samples were incubated overnight at 4°C with the following primary antibodies: Cdx2 (1:200, Cdx2-88, BioGenex Inc), Eomes (1:500,ab23345, Abcam), Tfap2c (1:300, sc-8977, Santa Cruz). After three washing steps, detection was performed with appropriate Alexa Fluor-conjugated antibodies (Invitrogen) at a 1:500 dilution in blocking buffer for 1 h at room temperature in the dark. After secondary antibody incubation, nuclei were stained with Hoechst (Sigma-Aldrich). Cells stained without primary antibodies served as controls. Western blotting was performed with 23 µg of cell lysates from TS cells cultured in TS and TX media by SDS-polycrylamide gel electrophoresis and blotted onto PVDF membranes. Membranes were blocked with 5% (v/v) BSA or 5% (v/v) non-fat milk powder in TBST (TBS with 0.1% Tween). Membranes were incubated with primary antibodies at room temperature for 2 h, washed three times with TBST and labeled with HRP conjugated antibodies (Dako) for 1 h at room temperature. After three washing steps with TBST, membranes were incubated in ECL substrate (Thermo Scientific). RNA isolation and analysis. RNA was isolated from cells using the RNeasy Minikit (Qiagen) according to the manufacturer's protocol. 500 ng total RNA was used for cDNA synthesis by RevertAidPremium (Thermo Scientific). For gene expression microarray analysis, RNA quality was tested using the Agilent 2100 Bioanalyzer (Agilent Technologies). 100 ng total RNA were used for cDNA synthesis. Biotin labeling and fragmentation were performed according to the Affymetrix user manual. cRNA was hybridized for 16h to Mouse Genome 430 2.0 GeneChip expression arrays (Affymetrix). After hybridization, arrays were washed and stained automatically on a GeneChip Fluidics station 450 (Affymetrix) and scanned with a GeneChip scanner 3000 7G (Affymetrix).
2014-01-28 | GSE47719 | GEO
Project description:3×Flag-atrA strain was grown in YEME and 50 mL were collected after 24 h. Cells were resuspended in phosphate buffer (pH=7.4) (PBS) and sonicated. After centrifugation at 12000 rpm, the supernatant was transferred and incubated with FLAG affinity gel (Yeasen) at 4 °C. The FLAG affinity gel was washed three times with 1 mL binding buffer (PBS contains 10% glycerol). After centrifugation at 2000 rpm, 500 μL PBS was added, and the interaction proteins were eluted after denaturation. WT strain was used as control. Protein identification and analysis was provided by Jingjie PTM BioLab (Hangzhou, China). Proteins were expressed in BL21 with the plasmids pET-28a-3×Flag-atrA and pET28a-clpX, respectively. The purified proteins were mixed and incubated at 30 °C for 3 h. The reaction system consisted of 4 μg AtrA, 4 μg ClpX and 3 mM ATP. The elution method is the same as for the pull-down assay after FLAG affinity gel purification at 4 °C. Samples were analyzed by western blot using anti-His mouse monoclonal antibody. AtrA was expressed in BL21 with the plasmid pET-32a-atrA and incubated with cell lysate of Δprc at 30 °C for 1 h. The protein processing and pupylation analysis were provided by Jingjie PTM BioLab (Hangzhou, China).
2024-05-23 | PXD037377 | Pride