Human Cell 293T BCAT1 IP GC-LC-MS
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture
SUBMITTER: 
Xiaochen Lu
LAB HEAD: Lei Qun-Ying
PROVIDER: PXD041556 | Pride | 2024-11-15
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| 20190505R11.raw | Raw | |||
| 20190505R12.raw | Raw | |||
| 20190505R13.raw | Raw | |||
| ExtendedTable4.xlsx | Xlsx | |||
| R14.raw | Raw |
Items per page: 5 1 - 5 of 7 |
Publications
Enhanced BCAT1 activity and BCAA metabolism promotes RhoC activity in cancer progression.
Qian Lin L Li Na N Lu Xiao-Chen XC Xu Midie M Liu Ying Y Li Kaiyue K Zhang Yi Y Hu Kewen K Qi Yu-Ting YT Yao Jun J Wu Ying-Li YL Wen Wenyu W Huang Shenglin S Chen Zheng-Jun ZJ Yin Miao M Lei Qun-Ying QY
Nature metabolism 20230619 7
Increased expression of branched-chain amino acid transaminase 1 or 2 (BCAT1 and BCAT2) has been associated with aggressive phenotypes of different cancers. Here we identify a gain of function of BCAT1 glutamic acid to alanine mutation at codon 61 (BCAT1<sup>E61A</sup>) enriched around 2.8% in clinical gastric cancer samples. We found that BCAT1<sup>E61A</sup> confers higher enzymatic activity to boost branched-chain amino acid (BCAA) catabolism, accelerate cell growth and motility and contribut ...[more]
Similar Datasets
Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.
2021-09-05 | GSE152862 | GEO
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:Ten 15 cm2 plates of HEK293 cells stably expressing TAP-vector, TAP-CSAG1, or TAP-CSAG2 were lysed with TAP lysis buffer (10% (v/v) glycerol, 50 mM HEPES-KOH pH 7.5, 100 mM KCl, 2 mM EDTA, 0.1% (v/v) NP-40, 10 mM NaF, 0.25 mM Na3VO4, 50 mM β-glyerolphosphate, 2 mM DTT, and 1X protease inhibitor cocktail (Sigma)) and cleared supernatants were bound to IgG-Sepharose beads (GE Amersham) and then washed in lysis buffer and TEV buffer (10 mM HEPES-KOH pH 8.0, 150 mM NaCl, 0.1% (v/v) NP-40, 0.5 mM EDTA, 1 mM DTT, and 1X protease inhibitor cocktail). Protein complexes were cleaved off the beads by TEV protease (Sigma) and incubated with calmodulin-Sepharose beads (GE Amersham) in calmodulin binding buffer (10 mM HEPES-KOH pH 8.0, 150 mM NaCl, 1 mM Mg acetate, 1 mM imidazole, 0.1% (v/v) NP-40, 6 mM CaCl2, 10 mM 2-mercaptoethanol) and then washed in calmodulin rinse buffer (50 mM Ammonium bicarbonate pH 8.0, 75 mM NaCl, 1 mM Mg Acetate, 1 mM Imidazole, 2 mM CaCl2) before eluted with SDS sample buffer, subjected to SDS-PAGE, and stained with GelCode Blue stain (Thermo Fisher Scientific) before protein identification by LC-MS/MS.
2020-09-01 | PXD020231 | Pride
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:BMDCs from WT, Fcgr2b-/- and Stinggt/gt were cultured, then stimulated with DMXAA for 3 and 6 hr. Cells were collected and lysed with 1 % IGEPAL CA-630, 0.5% TritonX-100, 150 mM NaCl, 50 mM Tris pH 7.4, 5% glycerol, 100 mM beta-Glycerophosphate, 2 mM Na3VO4 and 1X proteases inhibitor cocktail (Roche). First, antibody were mixed with the magnetic beads by adding 10 µg of STING antibody with 400 ug of SureBeads™ Protein A magnetic beads (Biorad, California, USA) and incubated for 1 hour at room temperature. Then, Protein lysates were added and incubated with antibody-conjugated beads for overnight at 4oC. After incubation, the beads were washed 3 times with wash buffer (150 mM NaCl and 50 mM Tris-HCL pH 7.4). Samples were eluted by adding 5X laemmli buffer and, boiled 950C for 10 minutes. The eluted protein samples were separated by 10 % SDS-PAGE gel. The STING interacting proteins from co-IP were analyzed by in-gel digestion followed by LC-MS/MS analysis.
2020-09-14 | PXD019239 | Pride
Project description:The CLIP procedures were conducted using HeLa cells according to the previous study (Liu et al., 2016) with slight modifications. Briefly, 4 plates of HeLa cells in 15 cm dishes were transfected with pPB/ALKBH3 plasmid for 48 h to reach about 90% cell confluency. After washing twice with ice-cold PBS, cells were irradiated twice with 400 mJ/cm2 at 254 nm by Stratalinker on ice. Cells were lysed in high salt lysis buffer (300 mM NaCl, 0.2% NP-40, 20 mM Tris-HCl PH 7.6, 0.5 mM DTT, protease inhibitor cocktail (1 tablet/50 ml), and RNase inhibitor (1:200)) at 4°C for 30 min. Supernatant was collected after centrifuged at 17,000 g at 4°C for 15 min and further treated with 1 U RNase T1 for 15 min at 24 °C. After centrifugation and filtration through 0.22 µM filter, 10% supernatant was collected as input for further sequencing analysis. Anti-Flag M2 beads (Sigma-Aldrich, St. Louis, MO, 80 μl) were washed three times with lysis buffer and incubated with the filtered supernatant at 4°C for 4 hrs. After removing the flow through by magnetic rack, beads and input were further incubated with 10 U RNase T1 exactly for 8 min. Samples were treated with 95 µL commercial PNK buffer and 0.5 U PNK for 15 min at 37 °C. After treatments, final concentration of 100 µM ATP and 1 U PNK were added and incubated at 37 °C for another 30 min. The RNA/protein complexes were eluted by NuPAGE 1 × loading buffer and fractionated by neutral NuPAGE 4–12% bis-tris gel. After cutting the gel, RNAs were recovered by proteinase K digestion in proteinase K buffer, followed by phenol/chloroform extraction using glycogen as carrier. The RNA fragments were ligated to adapter and established library by NEB small RNA library preparation kit (E7330S).
2022-03-01 | GSE112625 | GEO
Project description:Purpose: The goal of this study is to compare DAXX genome binding/occupancy by high throughput ChIP sequencing in control, wild type DAXX overexpressd cells (WT DAXX OE), and wild type DAXX SUMO interacting motif mutant overexpressd cells (DSM OE). Methods: The panel of MDA_MB-231 derived cell lines (control, WT DAXX OE, DSM OE) were cultured in complete DMEM. At about 90% confluency, the cells were crosslinked by adding 37 % formaldehyde to the final concentration of 1% for 10 min at room temperature. Crosslinking was stopped by adding glycine to the final concentration of 125 mM. Cells were lifted, washed with cold PBS, and pelleted by centrifugation. The cells were resuspended in a swelling buffer in the presence of the protease inhibitor cocktail (Sigma) and then pelleted and resuspended in the SDS lysis buffer. The lysates were transferred to a Covaris microTUBE and sonicated with an E220 Covaris Ultrasonicator. Chromatin fragmentation (~500 bps) was verified through agarose gel electrophoresis. The fragmented chromatins were diluted and incubated with a control IgG and the DAXX mAb (5G11) along with protein A/G magnetic beads. The beads were washed sequentially with a low salt buffer, high salt buffer, LiCl buffer, and TE buffer (twice). The immunoprecipitated chromatins were eluted at 65 °C for 15 min, and the eluted chromatins were subjected to proteinase K digestion at 65 °C for 3 h. The DNAs were recovered through a Qiagen mini-prep column. The immunoprecipitated DNAs were used for qPCR and library construction and high throughput sequencing using an Illumina Hi-Seq 2500 sequencer. Results: We surveyed genome-wide occupancy of DAXX using ChIP-seq technology. Overexpression of WT DAXX increased DAXX?s chromatin association specifically with lipogenic pathway genes where DSM OE reverse. Conclusions: Our study represents the first detailed analysis of DAXX occupancy in lipoegenic gene transciption.
2021-12-20 | GSE190783 | 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:gDNA was estracted and digested with cocktail of restrict enzymes MseI, DdeI, AluI, MboI, incubated at 37°C for 6 h. After purified by phenol/chloroform extraction, fragmented DNA was resuspended by TE, and quantified by Qubit 3.0 (Invitrogen). For each sample, 20 μg input DNA was immunoprecipitated overnight at 4°C in a shaker, with 1× DRIP binding buffer (10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) and 10 μg S9.6 antibody (ATCC, HB-8730). After adding 50 μl Dynabeads Protein G (Invitrogen, 10004D) and incubated for 4 h, the beads with antibody were washed by 1× DRIP binding buffer for 4 times at room temperature, each wash takes 10 min. Added 250 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS) and 5 U Proteinase K to beads/antibody complexes, incubated for 40 min in an Eppendorf ThermoMixer at 55°C, 1,000 rpm. Purified by phenol/chloroform extraction, and moved supernatant to a new tube, followed by adding 1/10 volume 3 M NaAc, 1 μl GlycoBlue (Invitrogen) and 1 volume isopropanol, precipitated at -20°C for 3 h and then centrifuged. After washed by 70% ethanol and dried, the DRIPed DNA pellet was resuspended by 75 μl low EDTA TE buffer (10 mM Tris-HCl 8.0, 0.1 mM EDTA). NGS libraries were constructed using Accel-NGS® 1S Plus DNA Library Kit (Swift Biosciences).
2019-10-15 | GSE134101 | GEO
Project description:Purpose: Investigation of DDX41 chromatin binding sites in U2OS cells Method: Expression of DDX41-GFP was induced for 48 hours before crosslinking using 1µg/ml docycycline. Control cells were not induced with doxycline. Cells were mildly crosslinked using 1% FA for 2 min at RT. Concanavalin A-coated beads were activated in Binding Buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1 mM MnCl2).1*10^6 U2OS cells were washed twice with Wash Buffer (Hepes-NaOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM protease inhibitor) at room temperature and afterwards immobilized on the activated beads in 1 ml Wash Buffer. Cells were permeabilized with 0.05% digitonin in Wash buffer for 4 min at RT. 1 µg Nanobody-GFP-MNase (in-house protein production, PMID:25362362) binding was performed at 4°C for 30 min in 0.05% digitonin-Wash buffer. After 2x washing, the MNase was activated by adding 3mM CaCl2 on ice for 30 min. 2x Stop Buffer (68 µl 5M NaCl, 40 µl 0.5 M EDTA, 20 µl 0.2 M EGTA, 10 µl 5% digitonin, 5 µl 10mg/ml RnaseA, )) was mixed with the samples to stop the reaction. Chromatin fragments were released by incubating the samples for 20 minutes at 37°C and centrifugation at 16.000×g for 10 minutes at 4°C. Supernatants were incubated at 70°C in the presence of 0.1% SDS and 5 µg proteinase K. Before library preparation, the DNA was recovered by phenol-chloroform extraction Results: We succesfully mapped DDX41 binding sites on cromatin in U2OS cells. DDX41 preferentially binds in the promoter region of genes.
2021-11-17 | GSE183083 | GEO